Human monoclonal antibody

ABSTRACT

The present invention provides an anti-CD81 antibody usable as a pharmaceutical product for human. Specifically, the present invention provides an anti-human CD81 antibody capable of binding to a peptide region consisting of the amino acid sequence of the amino acid numbers 80 to 175 in the amino acid sequence shown in SEQ ID NO:22.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is based on U.S. provisional patent applications No.61/420,136 filed on Dec. 6, 2010, the contents of which are incorporatedin full herein.

FIELD OF THE INVENTION

The present invention relates to a human monoclonal antibody molecule.Specifically, it relates to a human antibody molecule against human CD81and a pharmaceutical composition containing the molecule as an activeingredient.

BACKGROUND OF THE INVENTION

Bowels are organs which digest and absorb nutrients and water essentialfor activities of life of organisms. Meanwhile, they are also organswhich have an immunodefense performance for excluding foreign matterssuch as pathogens and keep life conservation by controllingcontradictory qualities in a well-balanced manner. It is however knownthat when the balance of these functions becomes abnormal, this dynamicequilibrium is broken to induce various bowel diseases. Especially,inflammatory bowel diseases (abbreviated as IBD), of which patients havebeen increased in number in recent years, are associated withabnormalities in digestive organs such as abdominal pain, diarrhea,mucous and bloody stool and the like and, in view of pathogenic statesthereof, grouped into ulcerative colitis and Crohn's disease.

Ulcerative colitis is a disease mainly showing diffuse bowel mucosalinflammation restricted to the large intestine, where repeatedinflammation leads to the onset of colorectal cancer, surgery is oftennecessary, and postoperative problems of increased frequency ofdefecation, stool leakage and onset of pouchitis are caused. Crohn'sdisease is a disease showing lesion spreading from the small intestineto the large intestine, and intense, discontinuous all layerinflammation starting from the submucosal layer, where repeatedinflammation leads to the intestinal complications (stenosis, fistula,abscess) that require operation (Inflamm. Bowel. Dis., 8, 244-250,2002).

In recent years, it has been known that an anti-TNF-α antibody iseffective as a therapeutic agent of Crohn's disease and ulcerativecolitis (N. Engl. J. Med., 353, 2462-2476, 2005). Also, an anti-α4integrin antibody Natalizumab has been reported to be effective as atherapeutic agent of Crohn's disease (J. Clin. Invest., 118, 825-826,2008). Nevertheless, in the current therapies including the antibodies,40-60% of IBD patients has not yet received a satisfactory medicaltreatment. Accordingly, the development of an effective therapeuticagent has been in high demand in a medical care (J. Clin.Gastroenterol., 41, 799-809, 2007).

CD81 is a cell surface molecule of 26 kDa, which is expressed inwide-ranging cells. It has an activity of decreasing a threshold of Bcell activation by forming a complex with CD21, CD19 and Leu 13 in a Bcell. In a T cell, it is associated with CD4 and CD8 to transducestimulatory signal into cells. In view of these matters, CD81 isconsidered to have a significant role in an immune response to aheterologous antigen. Moreover, it is involved in various integrinsphysiologically and functionally to activate VLA-4 (α4β1 integrin) in aB cell or LFA-1 (αLβ2 integrin) in a thymocyte.

As a disease associated with CD81, hepatitis C is known (Science, 282,938-941, 1998).

In recent years, it has been reported that anti-CD81 antibody is usefulfor the treatment of IBD (WO 2005/021792). IBD associated with T cellmigration (J. Clin. Invest., 118, 825-826, 2008; Inflamm. Bowel Dis.,16(4), 583-92, 2010; J. Pharmacol. Exp. Ther., 327(2), 383-92, 2008). Asother diseases associated with T cell migration, multiple sclerosis andpsoriasis are known (J. Clin. Invest., 118, 825-826, 2008; J.Neuroimmunol., 60, 17-28, 1995; Expert Opinion on Biological Therapy,3(2), 361-70, 2003).

To be specific, it has been reported that bowel mucosa layer T cells orperipheral blood T cells of a patient suffering from IBD such as Crohn'sdisease or ulcerative colitis highly express a chemokine receptor CXCR4and exhibit a strong chemotactic response to a chemokine CXCL12(Inflamm. Bowel Dis., 16(4), 583-92, 2010), and that colitis is cured byadministering a CXCR4 inhibitor to an IBD model, dextran sulfate-inducedmouse colitis model (J. Pharmacol. Exp. Ther., 327(2), 383-92, 2008),and that an anti-α-4 integrin antibody Natalizumab, which treats IBD bysuppressing T cell migration, has been approved as a pharmaceuticalproduct (J. Clin. Invest., 118, 825-826, 2008).

It has also been reported that T cell migration is important for thepathology of an animal model of multiple sclerosis, experimentalautoimmune encephalomyelitis (EAE) mouse (J. Neuroimmunol., 60, 17-28,1995). Natalizumab is thought to exert its therapeutic efficacy byblocking the α4 integrin-mediated adhesion of circulating T cells to theblood-brain barrier in EAE mice (J. Clin. Invest., 118, 825-826, 2008).Natalizumab is also effective for the treatment of multiple sclerosis.

Furthermore, it has been reported that T cells abundantly accumulate inpsoriatic skin and that an anti-LFA-1 antibody Efalizumab (trade name:Raptiva), which suppresses T cell migration, is effective for thetreatment of psoriasis (Expert Opinion on Biological Therapy, 3(2),361-70, 2003).

There arise various problems based on the species difference whenclinical application of an anti-CD81 antibody to human is desired. Forexample, administration of a mouse antibody to human may be limited byshort serum half-life, failure to trigger certain kinds of humaneffector function and induction of undesirable human immune response tothe mouse antibody (“human anti-mouse antibody” (HAMA) reaction) (Blood,62, 988-995, 1983; Cancer Res., 45, 879-885, 1985). Moreover, even ananti-TNFα antibody (Remicade), which is a chimeric molecule obtained bybinding the variable (V) region of a rodent antibody with the constant(C) region of a human antibody, may induce a human anti-chimericantibody (HACA) and cause an infusion reaction or loss of drug efficacy(Current Gastroenterology Reports, 5(6), 501-5, 2003).

SUMMARY OF THE INVENTION

Under the circumstances, an anti-CD81 antibody that can be used as apharmaceutical product is desired. However, such antibody is not known,and therefore, the problem to be solved by the present invention isprovision of an anti-CD81 antibody usable as a pharmaceutical productfor human.

In an attempt to solve the above-mentioned problem, the presentinventors have prepared fully human anti-CD81 antibodies from a humancomplementarity-determining region (CDR) library by a phage librarymethod, evaluated the region of human CD81 to which the antibodies bind,and found that antibodies bound to a certain region of CD81 showsuperior efficacy as well as high safety for human body, which resultedin the completion of the present invention. The present inventionprovides a human monoclonal antibody to human CD81. Furthermore, thepresent inventors obtained new findings that the anti-CD81 antibodiessuppressed T cell migration, which revealed that the antibody of thepresent invention was useful for the prophylaxis, improvement ortreatment of not only inflammatory bowel diseases such as Crohn'sdisease and ulcerative colitis but also diseases associated with T cellmigration such as multiple sclerosis and psoriasis. Furthermore, thepresent inventors found that the antibody of the present invention isnot only capable of merely binding to CD81-expressing cancer cells, butalso has a cytotoxic effect, due to its complement-dependentcytotoxicity (CDC), on cancer cells to which it has bound, and istherefore also useful in preventing, ameliorating or treating cancerscaused by CD81-expressing cancer cells, including hematological cancers(hematologic cancers, blood cancers, hematologic(al) malignancies).

Accordingly, the present invention is as follows.

[1] An anti-human CD81 antibody capable of binding to a peptide regionconsisting of the amino acid sequence of the amino acid numbers 80 to175 in the amino acid sequence shown in SEQ ID NO:22.[2] The antibody of [1], wherein the peptide region consists of theamino acid sequence of the amino acid numbers 113 to 175.[3] The antibody of [1] or [2], wherein the binding affinity of theantibody to at least one human CD81 variant selected from the groupconsisting of the following (1) to (13) is less than 40% of that to thehuman CD81 having the amino acid sequence shown in SEQ ID NO:22.(1) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein tyrosine at the amino acid number 127 is substituted withphenylalanine or tryptophan;(2) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein alanine at the amino acid number 130 is substituted withthreonine or valine;(3) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein valine at the amino acid number 135 is substituted with alanineor leucine;(4) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein aspartic acid at the amino acid number 137 is substituted withalanine or glutamic acid;(5) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein alanine at the amino acid number 143 is substituted withthreonine or valine;(6) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein histidine at the amino acid number 151 is substituted withalanine or arginine;(7) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein leucine at the amino acid number 154 is substituted with alanineor isoleucine;(8) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein glycine at the amino acid number 158 is substituted with alanineor serine;(9) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein alanine at the amino acid number 164 is substituted withthreonine or valine;(10) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein serine at the amino acid number 168 is substituted with alanineor threonine;(11) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein valine at the amino acid number 169 is substituted with alanineor leucine;(12) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein leucine at the amino acid number 170 is substituted with alanineor isoleucine; and(13) CD81 variant having the amino acid sequence shown in SEQ ID NO:22wherein asparagine at the amino acid number 172 is substituted withalanine or glutamine.[4] The antibody of any one of [1] to [3], wherein the binding affinityof the antibody to each of the above-identified human CD81 variants (9)and (11) is less than 40% of that to the human CD81 having the aminoacid sequence shown in SEQ ID NO:22.[5] An antibody having a binding property equivalent to that of theantibody of any one of [1] to [4], or binding to the human CD81 havingthe amino acid sequence shown in SEQ ID NO:22 competitively with theantibody of any one of [1] to [4].[6] An antibody binding to the human CD81 having the amino acid sequenceshown in SEQ ID NO:22 competitively with the antibody of any one of [1]to [4], and having a suppressive effect of T cell migration.[7] An anti-human CD81 antibody, which comprises all 6 CDRs described inany one of the following groups 1 to 24.

Group 1

(a-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 2

(a-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:37,(d-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-2) a CDR comprising the amino acid sequence shown in SEQ ID NO: 6

Group 3

(a-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:40,(d-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-3) a CDR comprising the amino acid sequence shown in SEQ ID NO: 6

Group 4

(a-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:43,(d-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 5

(a-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:46,(d-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 6

(a-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:49,(d-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 7

(a-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-7) a CDR comprising the amino acid sequence shown in SEQ ID NO: 2,(c-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:52,(d-7) a CDR comprising the amino acid sequence shown in SEQ ID NO: 4,(e-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 8

(a-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:43,(d-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:55

Group 9

(a-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:60,(b-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:61

Group 10

(a-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:66,(d-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 11

(a-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:69,and(f-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:70

Group 12

(a-12) a CDR comprising the amino acid sequence shown in SEQ ID NO: 60,(b-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:66,(d-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 13

(a-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:77,(e-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 14

(a-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:80,(b-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:81,and(f-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 15

(a-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-15) a CDR comprising the amino acid sequence shown in SEQ ID NO: 66,(d-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 16

(a-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:90,(d-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 17

(a-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:52,(d-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:93

Group 18

(a-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:98,(b-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:99

Group 19

(a-19) a CDR comprising the amino acid sequence shown in SEQ ID NO: 60,(b-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:99

Group 20

(a-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:90,(d-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 21

(a-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:55

Group 22

(a-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:66,(d-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:110,(e-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 23

(a-23) a CDR comprising the amino acid sequence shown in SEQ ID NO: 1,(b-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:115

Group 24

(a-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-24) a CDR comprising the amino acid sequence shown in SEQ ID (d-24) aCDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:120,and(f-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:6.[8] The antibody of [7], which comprises the combination of the lightchain variable region and the heavy chain variable region described inany one of the following groups 25 to 48.

Group 25

(g-1) a light chain variable region comprising the above-identified CDRs(a-1) to (c-1); and(h-1) a heavy chain variable region comprising the above-identified CDRs(d-1) to (f-1),

Group 26

(g-2) a light chain variable region comprising the above-identified CDRs(a-2) to (c-2); and(h-2) a heavy chain variable region comprising the above-identified CDRs(d-2) to (f-2),

Group 27

(g-3) a light chain variable region comprising the above-identified CDRs(a-3) to (c-3); and(h-3) a heavy chain variable region comprising the above-identified CDRs(d-3) to (f-3),

Group 28

(g-4) a light chain variable region comprising the above-identified CDRs(a-4) to (c-4); and(h-4) a heavy chain variable region comprising the above-identified CDRs(d-4) to (f-4),

Group 29

(g-5) a light chain variable region comprising the above-identified CDRs(a-5) to (c-5); and(h-5) a heavy chain variable region comprising the above-identified CDRs(d-5) to (f-5),

Group 30

(g-6) a light chain variable region comprising the above-identified CDRs(a-6) to (c-6); and(h-6) a heavy chain variable region comprising the above-identified CDRs(d-6) to (f-6),

Group 31

(g-7) a light chain variable region comprising the above-identified CDRs(a-7) to (c-7); and(h-7) a heavy chain variable region comprising the above-identified CDRs(d-7) to (f-7),

Group 32

(g-8) a light chain variable region comprising the above-identified CDRs(a-8) to (c-8); and(h-8) a heavy chain variable region comprising the above-identified CDRs(d-8) to (f-8),

Group 33

(g-9) a light chain variable region comprising the above-identified CDRs(a-9) to (c-9); and(h-9) a heavy chain variable region comprising the above-identified CDRs(d-9) to (f-9),

Group 34

(g-10) a light chain variable region comprising the above-identifiedCDRs (a-10) to (c-10); and(h-10) a heavy chain variable region comprising the above-identifiedCDRs (d-10) to (f-10),

Group 35

(g-11) a light chain variable region comprising the above-identifiedCDRs (a-11) to (c-11); and(h-11) a heavy chain variable region comprising the above-identifiedCDRs (d-11) to (f-11),

Group 36

(g-12) a light chain variable region comprising the above-identifiedCDRs (a-12) to (c-12); and(h-12) a heavy chain variable region comprising the above-identifiedCDRs (d-12) to (f-12),

Group 37

(g-13) a light chain variable region comprising the above-identifiedCDRs (a-13) to (c-13); and(h-13) a heavy chain variable region comprising the above-identifiedCDRs (d-13) to (f-13),

Group 38

(g-14) a light chain variable region comprising the above-identifiedCDRs (a-14) to (c-14); and(h-14) a heavy chain variable region comprising the above-identifiedCDRs (d-14) to (f-14),

Group 39

(g-15) a light chain variable region comprising the above-identifiedCDRs (a-15) to (c-15); and(h-15) a heavy chain variable region comprising the above-identifiedCDRs (d-15) to (f-15),

Group 40

(g-16) a light chain variable region comprising the above-identifiedCDRs (a-16) to (c-16); and(h-16) a heavy chain variable region comprising the above-identifiedCDRs (d-16) to (f-16),

Group 41

(g-17) a light chain variable region comprising the above-identifiedCDRs (a-17) to (c-17); and(h-17) a heavy chain variable region comprising the above-identifiedCDRs (d-17) to (f-17),

Group 42

(g-18) a light chain variable region comprising the above-identifiedCDRs (a-18) to (c-18); and(h-18) a heavy chain variable region comprising the above-identifiedCDRs (d-18) to (f-18),

Group 43

(g-19) a light chain variable region comprising the above-identifiedCDRs (a-19) to (c-19); and(h-19) a heavy chain variable region comprising the above-identifiedCDRs (d-19) to (f-19),

Group 44

(g-20) a light chain variable region comprising the above-identifiedCDRs (a-20) to (c-20); and(h-20) a heavy chain variable region comprising the above-identifiedCDRs (d-20) to (f-20),

Group 45

(g-21) a light chain variable region comprising the above-identifiedCDRs (a-21) to (c-21); and(h-21) a heavy chain variable region comprising the above-identifiedCDRs (d-21) to (f-21),

Group 46

(g-22) a light chain variable region comprising the above-identifiedCDRs (a-22) to (c-22); and(h-22) a heavy chain variable region comprising the above-identifiedCDRs (d-22) to (f-22),

Group 47

(g-23) a light chain variable region comprising the above-identifiedCDRs (a-23) to (c-23); and(h-23) a heavy chain variable region comprising the above-identifiedCDRs (d-23) to (f-23),

Group 48

(g-24) a light chain variable region comprising the above-identifiedCDRs (a-24) to (c-24); and(h-24) a heavy chain variable region comprising the above-identifiedCDRs (d-24) to (f-24).[9] The antibody of [8], which comprises the combination of the lightchain variable region and the heavy chain variable region described inany one of the following groups 49 to 72.

Group 49

(i-1) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:8; and(j-1) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 50

(i-2) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:38; and(j-2) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 51

(i-3) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:41; and(j-3) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 52

(i-4) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:44; and(j-4) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 53

(i-5) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:47; and(j-5) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 54

(i-6) a light chain variable region comprising amino acid sequence shownin SEQ ID NO:50; and(j-6) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 55

(i-7) a light chain variable region comprising amino acid sequence shownin SEQ ID NO:53; and(j-7) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:10,

Group 56

(i-8) a light chain variable region comprising amino acid sequence shownin SEQ ID NO:56; and(j-8) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:57,

Group 57

(i-9) a light chain variable region comprising amino acid sequence shownin SEQ ID NO:62; and(j-9) a heavy chain variable region comprising amino acid sequence shownin SEQ ID NO:63,

Group 58

(i-10) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:67; and(j-10) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10,

Group 59

(i-11) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:71; and(j-11) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:72,

Group 60

(i-12) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:75; and(j-12) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10,

Group 61

(i-13) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:8; and(j-13) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:78,

Group 62

(i-14) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:82; and(j-14) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:83,

Group 63

(i-15) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:86; and(j-15) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:87,

Group 64

(i-16) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:91; and(j-16) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10,

Group 65

(i-17) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:94; and(j-17) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:95,

Group 66

(i-18) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:100; and(j-18) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:101,

Group 67

(i-19) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:104; and(j-19) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:101,

Group 68

(i-20) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:106; and(j-20) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10,

Group 69

(i-21) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:108; and(j-21) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:57,

Group 70

(i-22) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:111; and(j-22) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:112,

Group 71

(i-23) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:116; and(j-23) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:117,

Group 72

(i-24) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:121; and(j-24) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:122.[10] The antibody of [8] or [9], which comprises the combination of thelight chain and the heavy chain described in any one of the followinggroups 73 to 96.

Group 73

(k-1) a light chain comprising the amino acid sequence shown in SEQ IDNO:26; and(l-1) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 74

(k-2) a light chain comprising the amino acid sequence shown in SEQ IDNO:39; and(l-2) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 75

(k-3) a light chain comprising the amino acid sequence shown in SEQ IDNO:42; and(l-3) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 76

(k-4) a light chain comprising the amino acid sequence shown in SEQ IDNO:45; and(l-4) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 77

(k-5) a light chain comprising the amino acid sequence shown in SEQ IDNO:48; and(l-5) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 78

(k-6) a light chain comprising the amino acid sequence shown in SEQ IDNO:51; and(l-6) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 79

(k-7) a light chain comprising the amino acid sequence shown in SEQ IDNO:54; and(l-7) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 80

(k-8) a light chain comprising the amino acid sequence shown in SEQ IDNO:58; and(l-8) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:59,

Group 81

(k-9) a light chain comprising the amino acid sequence shown in SEQ IDNO:64; and(l-9) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:65,

Group 82

(k-10) a light chain comprising the amino acid sequence shown in SEQ IDNO:68; and(l-10) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 83

(k-11) a light chain comprising the amino acid sequence shown in SEQ IDNO:73; and(l-11) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:74,

Group 84

(k-12) a light chain comprising the amino acid sequence shown in SEQ IDNO:76; and(l-12) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 85

(k-13) a light chain comprising the amino acid sequence shown in SEQ IDNO:26; and(l-13) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:79,

Group 86

(k-14) a light chain comprising the amino acid sequence shown in SEQ IDNO:84; and(l-14) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:85,

Group 87

(k-15) a light chain comprising the amino acid sequence shown in SEQ IDNO:88; and(l-15) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:89,

Group 88

(k-16) a light chain comprising the amino acid sequence shown in SEQ IDNO:92; and(l-16) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 89

(k-17) a light chain comprising the amino acid sequence shown in SEQ IDNO:96; and(l-17) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:97,

Group 90

(k-18) a light chain comprising the amino acid sequence shown in SEQ IDNO:102; and(l-18) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:103,

Group 91

(k-19) a light chain comprising the amino acid sequence shown in SEQ IDNO:105; and(l-19) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:103,

Group 92

(k-20) a light chain comprising the amino acid sequence shown in SEQ IDNO:107; and(l-20) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28,

Group 93

(k-21) a light chain comprising the amino acid sequence shown in SEQ IDNO:109; and(l-21) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:59,

Group 94

(k-22) a light chain comprising the amino acid sequence shown in SEQ IDNO:113; and(l-22) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:114,

Group 95

(k-23) a light chain comprising the amino acid sequence shown in SEQ IDNO:118; and(l-23) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:119,

Group 96

(k-24) a light chain comprising the amino acid sequence shown in SEQ IDNO:123; and(l-24) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:124.[11] An antibody binding to the human CD81 having the amino acidsequence shown in SEQ ID NO:22 competitively with the antibody of anyone of [7] to [10].[12] The antibody of [11], which has a suppressive effect of T cellmigration.[13] An anti-human CD81 antibody, wherein the antibody comprises one ormore of CDRs described in any one of the groups 1 to 24 in [7] and bindsto the human CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with the antibody comprising all 6 CDRs described in saidgroup.[14] An anti-human CD81 antibody, wherein the antibody has a 90%sequence homology with any one of antibodies of [7] and binds to thehuman CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with said antibody.[15] An anti-human CD81 antibody comprising:(a-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:11;(b-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:12;(c-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:13;(d-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:14;(e-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:15;and(f-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:16.[16] The antibody of [15], which comprises:(g-25) a light chain variable region comprising the above-identifiedCDRs (a-25) to (c-25); and(h-25) a heavy chain variable region comprising the above-identifiedCDRs (d-25) to (f-25).[17] The antibody of [16], which comprises:(i-25) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:18; and(j-25) a heavy chain variable region comprising the amino acid sequenceshown in SEQ ID NO:20.[18] The antibody of [16] or [17], which comprises:(k-25) a light chain comprising the amino acid sequence shown in SEQ IDNO:30; and(l-25) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:32[19] An antibody binding to the human CD81 having the amino acidsequence shown in SEQ ID NO:22 competitively with the antibody of anyone of [15] to [18].[20] The antibody of [19], which has a suppressive effect of T cellmigration.[21] An anti-human CD81 antibody, wherein the antibody comprises one ormore of CDRs in [15] and binds to the human CD81 having the amino acidsequence shown in SEQ ID NO:22 competitively with the antibody describedin [15].[22] An anti-human CD81 antibody, wherein the antibody has a 90%sequence homology with the antibody described in [15] and binds to thehuman CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with said antibody.[23] The antibody of any one of [1] to [6], [11] to [14] and [19] to[22] which is a humanized or human antibody.[24] The antibody of any one of [7] to [10] and [15] to [18] which is ahumanized or human antibody.[25] The polynucleotide comprising a nucleotide sequence that encodes aheavy chain variable region and a light chain variable region of theantibody of any one of [1] to [24].[26] A combination of the polynucleotide comprising a nucleotidesequence that encodes a heavy chain variable region of the antibody ofany one of [1] to [6], [11] to [14] and [19] to [23], and thepolynucleotide comprising a nucleotide sequence that encodes a lightchain variable region of the antibody of any one of [1] to [6], [11] to[14] and [19] to [23].[27] A combination of the polynucleotide comprising a nucleotidesequence that encodes a heavy chain variable region of the antibody ofany one of [7] to [10], [15] to [18] and [24], and the polynucleotidecomprising a nucleotide sequence that encodes the corresponding lightchain variable region of the antibody of any one of [7] to [10], [15] to[18] and [24].[28] The polynucleotide comprising a nucleotide sequence that encodes aheavy chain and a light chain of the antibody of any one of [1] to [24].[29] A combination of the polynucleotide comprising a nucleotidesequence that encodes a heavy chain of the antibody of any one of [1] to[6], [11] to [14] and [19] to [23], and the polynucleotide comprising anucleotide sequence that encodes a light chain of the antibody of anyone of [1] to [6], [11] to [14] and [19] to [23].[30] A combination of the polynucleotide comprising a nucleotidesequence that encodes a heavy chain of the antibody of any one of [7] to[10], [15] to [18] and [24], and the polynucleotide comprising anucleotide sequence that encodes the corresponding light chain of theantibody of any one of [7] to [10], [15] to [18] and [24].[31] An expression vector comprising the polynucleotide of [25] or [28].[32] A recombinant cell transformed with the expression vector of [31].[33] A recombinant cell transformed with the expression vectorcomprising the polynucleotide comprising a nucleotide sequence thatencodes a heavy chain of the antibody of any one of [1] to [6], [11] to[14] and [19] to [23], and with the expression vector comprising thepolynucleotide comprising a nucleotide sequence that encodes a lightchain of the antibody of any one of [1] to [6], [11] to [14] and [19] to[23].[34] A recombinant cell transformed with the expression vectorcomprising the polynucleotide comprising a nucleotide sequence thatencodes the heavy chain of the antibody of any one of [7] to [10], [15]to [18] and [24], and with the expression vector comprising thepolynucleotide comprising a nucleotide sequence that encodes thecorresponding light chain of the antibody of any one of [7] to [10],[15] to [18] and [24].[35] A method of producing an anti-human CD81 antibody, comprisingculturing the recombinant cell of any one of [32] to [34], andrecovering the antibody from the culture obtained.[36] A pharmaceutical composition comprising the antibody of any one of[1] to [24].[37] An agent for the prophylaxis, improvement or treatment of a diseaseselected from inflammatory bowel disease, multiple sclerosis, psoriasisand hematological cancer comprising the antibody of any one of [1] to[24].[38] The antibody of any one of [1] to [3], wherein the binding affinityof the antibody to each of the above-identified human CD81 variants (3),(4), (8), (11) and (12) is less than 40% of that to the human CD81having the amino acid sequence shown in SEQ ID NO:22.[39] The antibody of any one of [1] to [3], wherein the binding affinityof the antibody to each of the above-identified human CD81 variants (3),(4), (6) and (8) to (13) is less than 40% of that to the human CD81having the amino acid sequence shown in SEQ ID NO:22.[40] The antibody of any one of [1] to [3], wherein the binding affinityof the antibody to each of the above-identified human CD81 variants (1)to (5), (7), (8), (11), and (12) is less than 40% of that to the humanCD81 having the amino acid sequence shown in SEQ ID NO:22.

The present invention can provide a human monoclonal antibody againsthuman CD81 with a superior drug efficacy and low immunogenicity tohuman. Since the present inventors obtained new findings that theanti-CD81 antibodies suppressed T cell migration, and moreover,exhibited cytotoxic effect on cancer cells, the antibody of the presentinvention is also useful for the prophylaxis, improvement or treatmentof inflammatory diseases including inflammatory bowel diseases, anddiseases associated with T cell migration including autoimmune diseasessuch as multiple sclerosis and psoriasis, as well as cancers caused byCD81-expressing cancer cells, including hematological cancers.

DETAILED DESCRIPTION OF THE INVENTION

In the present specification, the indication using abbreviations such asamino acid, (poly)peptide, (poly)nucleotide and the like follows thedefinitions of IUPAC-IUB [IUPAC-IUB Communication on BiologicalNomenclature, Eur. J. Biochem., 138: 9 (1984)], “Guideline for preparingspecification and the like containing nucleotide sequence or amino acidsequence” (ed. Japan Patent Office), and conventional marks used in thefield.

Herein, the “gene” or “DNA” is used in the meaning that it includes notonly a double-stranded DNA but also respective single-stranded DNAs, asense strand and an antisense strand constituting the double-strandedDNA. It is not particularly limited by the length thereof. Accordingly,the gene (DNA) in the specification includes, unless otherwiseinstructed, a double-stranded DNA including a human genomic DNA, asingle-stranded DNA (plus strand) including a cDNA, a single-strandedDNA having a sequence complementary to the plus strand (complementarystrand) and fragments thereof.

Herein, the term “CD81 gene” means a human CD81 gene (DNA) shown by SEQID NO:21, or naturally occurring mutants or polymorphic variants thereof(except those encoding any of the mutant proteins of (1) to (13)described below, as a result of the mutation or polymorphism). Suchmutants or polymorphic variants include, for example, those registeredin the SNP database available form NCBI.

Herein, the term “CD81 protein” or simply “CD81” means a human CD81protein shown by SEQ ID NO:22, or a protein encoded by the naturallyoccurring mutant or polymorphic variant DNAs mentioned above.

The “antibody” used herein encompasses a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a single-stranded antibody, ora part thereof capable of binding with its antigen such as an Fabfragment or a fragment generated from an Fab expression library.

Herein, the term “epitope” is a region of an antigen to which anantibody binds. In certain embodiments, it includes any site on anantigen that is capable of specific binding to an immunoglobulin or Tcell receptor or B cell receptor. Antigen determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. In certainembodiments, it can be mentioned that an antibody specifically binds toits target antigen when it preferentially recognizes the antigen in acomplex mixture of proteins and/or macromolecules.

Structure of Antibody

The basic structure of an antibody molecule is shared by all classes,and consists of both a heavy chain having a molecular weight of 50000 to70000 and a light chain having a molecular weight of 20000 to 30000(Immunology 4th ed., I. Roitt, J. Brostoff, D. Male eds., Mosby-YearBook, 1996). A heavy chain usually consists of a polypeptide chaincomprising about 440 amino acids; heavy chains have characteristicstructures in each different classes, and are called the γ, μ, α, δ, andε chains corresponding to IgG, IgM, IgA, IgD, and IgE. Furthermore, IgGoccurs as IgG1, IgG2, IgG3, and IgG4, and corresponding chains arecalled γ1, γ2, γ3, and γ4, respectively. A light chain usually consistsof a polypeptide chain comprising about 220 amino acids; two types, typeL and type K, are known, and are called the λ, and κ chains,respectively. Regarding the peptide configuration of the basic structureof an antibody molecule, two homologous heavy chains and two homologouslight chains are bound via disulfide bonds (S—S bonds) and non-covalentbonds, and the molecular weight is 150000 to 190000. The two kinds oflight chains are capable of paring with any heavy chain. Each antibodymolecule always consists of two identical light chains and two identicalheavy chains.

There are four intra-molecular S—S bonds in a heavy chain (five bondsfor μ and ε chains) and two in a light chain; one loop is formed per 100to 110 amino acid residues, and this steric structure is alike among theloops, and is called a structural unit or domain. For both heavy chainsand light chains, the amino acid sequence of the domain located at the Nterminus thereof is inconstant, even in preparations from the same class(subclass) of the same animal species, and this domain is called avariable region (V region) (the heavy chain variable region domain iscalled as V_(H) and the light chain variable region domain is called asV_(L)). The amino acid sequence on the C-terminal side therefrom isnearly constant in each class or subclass, and is called a constantregion (C region) (the domains are expressed as C_(H)1, C_(H)2, C_(H)3and C_(L), respectively).

The antigen determination site of an antibody consists of V_(H) andV_(L), and the binding specificity depends on the amino acid sequence ofthis site. On the other hand, biological activities such as binding tocomplements or various cells reflect the differences in C regionstructure among the various classes of Ig. The variability of thevariable regions of light chain and heavy chain has been found to benearly limited to three small hypervariable regions existing in bothchains, and these regions are called complementarity determining region(CDR). Several numbering systems for identifying CDRs are in common use.The Kabat definition is based on sequence variability, and the Chothiadefinition is based on the location of the structural loop regions. TheAbM definition is a compromise between the Kabat and Chothia approaches.The CDRs of the light chain and heavy chain variable regions are boundedaccording to the Kabat, Chothia, or AbM algorithm (Martin et al. (1989)Proc. Natl. Acad. Sci. USA 86: 9268-9272; Martin et al. (1991) MethodsEnzymol. 203: 121-153; Pedersen et al. (1992) Immunomethods 1: 126; andRees et al. (1996) In Sternberg M. J. E. (ed.), Protein StructurePrediction, Oxford University Press, Oxford, pp. 141-172).

In the case of 002-A07 antibody, the CDRs in the heavy chain variableregion are bounded by the residues at amino acid Nos. 29-42 (CDR1-H),49-67 (CDR2-H) and 97-108 (CDR3-H) of the amino acid sequence shown bySEQ ID NO:10, and the CDRs in the light chain variable region arebounded by the residues at amino acid Nos. 22-36 (CDR1-L), 52-58(CDR2-L) and 90-101 (CDR3-L) of the amino acid sequence shown by SEQ IDNO:8. In the case of 005-C01 antibody, the CDRs in the heavy chainvariable region are bounded by the residues at amino acid Nos. 29-42(CDR1-H), 49-67 (CDR2-H) and 97-102 (CDR3-H) of the amino acid sequenceshown by SEQ ID NO:20, and the CDRs in the light chain variable regionare bounded by the residues at amino acid Nos. 22-35 (CDR1-L), 51-57(CDR2-L) and 89-99 (CDR3-L) of the amino acid sequence shown by SEQ IDNO:18.

The portion other than CDRs of the variable region is called a frameworkregion (FR), and is relatively constant. The framework region employs aβ sheet conformation, and CDRs can form a loop connecting the β sheetstructure. CDRs in each chain are maintained in the three dimensionalstructure thereof by the framework regions and form an antigen bindingsite together with CDRs from the other chain.

Binding Assay of Antibody

Antibody binding can be confirmed by any known assay method, such asdirect and indirect sandwich assays, flow cytometry andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc., 1987, pp. 147-158). In the presentinvention, the binding of an anti-human CD81 monoclonal antibody with ahuman CD81 polypeptide or a cell presenting same on its surface can bemeasured, for example, by the following method.

As a typical method, exemplified is a method comprising adsorbing ahuman CD81 polypeptide (antigen) onto a solid phase, blocking the solidphase with a protein that is not involved in the subsequentantigen-antibody reaction or enzyme reaction (e.g., skim milk, albuminetc.), contacting and incubating a human anti-human CD81 monoclonalantibody (test antibody) with the solid phase, removing an unreactedantibody by B/F separation, and adding a labeled secondary antibodyspecifically reacting with the test antibody (e.g., anti-human IgG,etc.) to the solid phase to determine the amount of the label on thesolid phase. As the solid phase, for example, insoluble polysaccharidessuch as agarose, dextran and cellulose, synthetic resins such asplastic, polystylene, polyacrylamide and silicone (e.g., tube,microplate, etc.), or glass (beads, tube, etc.) can be used. Themembrane fraction of a cell expressing a human CD81 polypeptide may beused as an antigen to be adsorbed onto the solid phase, as shown inExperimental Example 7(2) described below. As a means forimmobilization, an antigen may be recombinantly expressed as a fusionprotein with a peptide (e.g., His-tag, GST, MBP, etc.) capable ofbinding with a solid phase (Ni-, glutathione-, maltose-carrier, etc.)and bound to the solid phase with affinity thereto, as shown inExperimental Example 7(2). As the labeling agent, radioisotopes,enzymes, fluorescent substances, luminescent substances and the like canbe used. As examples of the radioisotopes, [¹²⁵I], [¹³¹], [³H], [¹⁴C]and the like can be mentioned. As examples of the enzymes, stableenzymes with high specific activity are preferred; for example,β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase and the like can be mentioned. As examples of thefluorescent substances, fluorescamine, fluorescein isothiocyanate andthe like can be mentioned. As examples of the luminescent substances,luminol, luminol derivatives, luciferin, luciferin and the like can bementioned.

In the sandwich method, an immobilized anti-human CD81 antibody, whichbinds to human CD81 at a site different from the site to which theantibody of the present invention binds, is reacted with a human CD81polypeptide (antigen), and further reacted with an anti-human CD81monoclonal antibody (test antibody). After removing an unreactedantibody by B/F separation, a labeled secondary antibody specificallyreacting with the test antibody (e.g., anti-human IgG, etc.) is added todetermine the amount of the label on the solid phase. The labeling agentand the solid phase may be the same as mentioned above.

To examine binding of anti-human CD81 monoclonal antibodies to livecells expressing a human. CD81 polypeptide, flow cytometry can be used.Briefly, a cell expressing human CD81 (grown under standard growthconditions) can be mixed with an anti-human CD81 antibody (testantibody) in, for example, PBS containing 0.1% BSA, and incubated at 37°C. for 1 hour. After washing, the cells are reacted with a secondaryantibody (e.g., anti-human IgG antibody) labeled with a fluorescent suchas fluorescein or phycoerythrin (PE) under the same conditions as thereaction with test antibody. The samples can be analyzed by a flowcytometer using light and side scatter properties to gate on singlecells. For example, when the percentage of the cells possessing afluorescent intensity greater than that when non-specific antibody(e.g., human IgG) is used is 90% or more, preferably 95% or more, morepreferably 97% or more, the test antibody can be confirmed tospecifically bind to the antigen. An alternative assay usingfluorescence microscopy may be used in addition to or instead of theflow cytometry assay. Cells can be stained exactly as described aboveand examined by fluorescence microscopy. This method allowsvisualization of individual cells.

Competitive Assay

Competitive assays such as competitive ELISA can be used to determinethe binding constant (Ka) of an anti-human CD81 antibody or identifyanother antibody of the present invention, which binds to a human CD81competitively with the antibody of the present invention alreadyobtained (known antibody). The competitive assays are carried out byadding a free antigen or known antibody to the reaction system of thesolid phase and the test antibody in the binding assay usingantigen-immobilized solid phase mentioned above. For example, a givenconcentration of test antibody solution and mixtures in which variousconcentrations of antigen are added to the test antibody solution arecontacted and incubated with an antigen-immobilized solid phase,respectively, and the amounts of label on the respective solid phasesare measured. The binding constant can be calculated as the gradient ofgraph showing the results of Scatchard analysis based on the measuredvalues for respective antigen concentrations. On the other hand, anantibody binding to a human CD81 competitively with the antibody of thepresent invention can be identified by reacting a labeled known antibody(the inventive antibody) and various concentrations of test antibodywith the antigen-immobilized solid phase, and selecting the testantibody that reduced the amount of label on the solid phase in adose-dependent manner.

Antibody of the Present Invention

The antibody of the present invention is an anti-human CD81 antibodycapable of binding to a peptide region consisting of the amino acidsequence of the amino acid numbers 80 to 175, preferably 113 to 175, inthe amino acid sequence shown in SEQ ID NO:22, or an antibody binding tothe human CD81 consisting of the amino acid sequence shown in SEQ IDNO:22 competitively with said anti-human CD81 antibody. The amino acidsequence shown in SEQ ID NO:22 is a reported amino acid sequence ofhuman CD81 protein (EMBO J., 20: 12-18, 2001), and registered in NCBIdatabase as Refseq ID: NP_(—)004347. Hereinafter, the protein consistingof the amino acid sequence is also referred to as “naturally occurringhuman CD81”, “wild type CD81” or “wild type human CD81”.

The present invention is based on the findings that anti-human CD81antibodies that recognize the peptide region mentioned above as anepitope exert their therapeutic effect such as suppression of T cellmigration with no or few side effects.

The antibody of the present invention can be any monoclonal antibody, aslong as it binds to the particular peptide region of the wild type humanCD81 mentioned above, or binds to the wild type human CD81 competitivelywith an antibody binding to the peptide region.

Epitope Analysis

Epitope analysis can be carried out according to various known methods(Epitope Mapping Protocols/Second Edition, Mike Schutkowski, UlrichReineke, Ann N Y Acad. Sci. 2010 January; 1183: 267-87). More detailedepitope analysis for an antibody can be performed by binding inhibitionassay, homolog scanning and/or alanine scanning (for example, see JP2009-159948 A, Science, 244: 1081-1085 (1989)).

Alanine scanning is a method to determine whether each amino acidresidue of human CD81 is necessary for binding of its antibody theretoby preparing mutants wherein each of the amino acid residues of wildtype human CD81 is substituted with alanine, and examining differencesin binding activity of the antibody between against CD81 mutants andagainst wild type human CD81.

Homolog scanning is a method to determine which amino acid residues canbe inserted, substituted or deleted without adverse effect on activityby substituting at least one amino acid residue of wild type human CD81polypeptide with other homologous amino acid(s). In this method, theamino acid sequence of human CD81 polypeptide is compared to those ofknown homologous protein molecules and the number of amino acid changesgenerated within the region having a high homology is minimized (ProteinScience, 14, 2405-2413 (2005)).

The homolog scanning and alanine scanning mutagenesis carried out in thepresent invention (see Experimental examples 8 and 9 etc.) has revealedthat the substitutions of the particular amino acid residues in thepeptide region mentioned above with other amino acids remarkably reducethe binding affinity of the anti-human CD81 antibodies to the human CD81mutants. These findings show that the substituted amino acid residuesmainly contribute to the binding of the antibodies to wild type humanCD81.

Accordingly, in a preferable embodiment, the antibody of the presentinvention is characterized in that its binding affinity to at least oneCD81 variant selected from the following (1) to (13) is less than 40%when its binding affinity to the above-mentioned wild type human 0081 is100%:

-   -   (1) 0081 variant in which the 127th tyrosine (Y) of wild type        human CD81 is substituted with phenylalanine or tryptophan;    -   (2) 0081 variant in which the 130th alanine (A) of wild type        human CD81 is substituted with threonine or valine;    -   (3) CD81 variant in which the 135th valine (V) of wild type        human CD81 is substituted with alanine or leucine;    -   (4) CD81 variant in which the 137th aspartic acid (D) of wild        type human CD81 is substituted with alanine or glutamic acid;    -   (5) CD81 variant in which the 143rd alanine (A) of wild type        human CD81 is substituted with threonine or valine;    -   (6) CD81 variant in which the 151st histidine (H) of wild type        human CD81 is substituted with alanine or arginine;    -   (7) CD81 variant in which the 154th leucine (L) of wild type        human CD81 is substituted with alanine or isoleucine;    -   (8) CD81 variant in which the 158th glycine (G) of wild type        human CD81 is substituted with alanine or serine;    -   (9) CD81 variant in which the 164th alanine (A) of wild type        human CD81 is substituted with threonine or valine;    -   (10) CD81 variant in which the 168th serine (S) of wild type        human CD81 is substituted with alanine or threonine;    -   (11) CD81 variant in which the 169th valine (V) of wild type        human CD81 is substituted with alanine or leucine;    -   (12) CD81 variant in which the 170th leucine (L) of wild type        human CD81 is substituted with alanine or isoleucine; and    -   (13) CD81 variant in which the 172nd asparagine (N) of wild type        human CD81 is substituted with alanine or glutamine;        wherein the positions of the amino acid residues to be        substituted are identified as the amino acid numbers of the        amino acid sequence shown in SEQ ID NO:22.

Here, the above-mentioned CD81 variants can be produced by thedescription of the following Experimental Example or a known method, forexample, introducing a suitable nucleotide change causing an amino acidsubstitution into a DNA encoding the wild type human CD81 polypeptide,or chemically synthesizing a desired variant polypeptide. The mutationof human CD81 polypeptide described here can be formed by, for example,the technique or guideline relating to the preservative ornon-preservative mutation shown in U.S. Pat. No. 5,364,934 (for example,see Experimental examples 8 and 9 of the present invention).

The binding affinity of an antibody to the wild type or a mutant humanCD81 can be determined by various binding assays described above. Thebinding constant (Ka) of the antibody of the present invention againstwild type human CD81 is at least 1×10⁷ M⁻¹, preferably 10⁸ M⁻¹, morepreferably 10⁸ M⁻¹, even more preferably 10¹⁰ M⁻¹. The binding constantof the antibody can be determined by the competitive assay describedabove or other well-known methods such as surface plasmon resonance(SPR).

More preferably, the antibody of the present invention is such that thebinding affinity to each of the human CD81 variants (9) and (11) aboveis less than 40% of that to wild type human CD81. The antibody of thepresent invention is also such that the binding affinity to each of thehuman 0081 variants (3), (4), (9) and (11) above is less than 40% ofthat to wild type human 0081, that the binding affinity to each of thehuman CD81 variants (3), (4), (9), (10) and (11) above is less than 40%,that the binding affinity to each of the human CD81 variants (3), (4),(8), (9), (10) and (11) above is less than 40%, or the binding affinityto each of the human CD81 variants (3), (4), (6), (8), (9), (10), (11)and (12) above is less than 40%. The series of antibodies possessing thebinding characteristic are described in detail in (i) below. In the caseof an antibody comprising all six CDRs belonging to the group 1, whichrepresents a specific mode of embodiment of the present inventiondescribed in (i) below, the binding affinity to each of the human CD81variants (3), (4), (6) and (8) to (13) above is less than 40%.

In another preferred embodiment, the antibody of the present inventionis such that the binding affinity to each of the human CD81 variants (1)to (5), (7), (8), (11) and (12) above is less than 40% of that to wildtype human CD81. The series of antibodies possessing the bindingcharacteristic are described in detail in (ii) below. Human CD81variants shared by this antibody and the aforementioned antibody in thespecific mode of embodiment described in (i) below, whose bindingaffinity is less than 40% of that to wild type human CD81, are the humanCD81 variants (3), (4), (8), (11) and (12) above, respectively.

The present invention also provides an antibody binding to wild typehuman CD81 competitively with any antibody of the present inventiondescribed above. The antibody can bind to wild type human CD81 antibodyin a region containing amino acid(s) outside the amino acid region ofamino acid Nos. 80-175 in the amino acid sequence shown in SEQ ID NO:22,as long as it has a suppressive effect on T cell migration and/or acytotoxic effect on CD81-expressing cancer cells. While it is desirablethat the suppressive effect on T cell migration and the cytotoxicactivity on CD81-expressing cancer cells be equivalent (e.g., 0.5-2fold) to those of an antibody binding to wild type human CD81 in theamino acid region of amino acid Nos. 80-175 or Nos. 113-175 in the aminoacid sequence shown in SEQ ID NO:22, the extent of the activity may bedifferent, as long as the antibody exerts a prophylactic, amelioratingor therapeutic effect on inflammatory bowel diseases, multiple sclerosispsoriasis, or hematological cancers of humans. The “competitive binding”of antibodies to their antigen can be examined by the competitive assaydescribed above.

(i) One Specific Embodiment

One preferable embodiment is an antibody having a binding affinity ofless than 40% to each of the human CD81 variants described in (9) and(11) when its binding affinity to wild type human CD81 is 100%.

A preferable example of the antibodies having the binding propertiesmentioned above is:

Group 1

(a-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-1) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 2

(a-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:37,(d-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-2) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 3

(a-3) a CDR comprising the amino acid sequence shown in SEQ ID NO: 1,(b-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:40,(d-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-3) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 4

(a-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:43,(d-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-4) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 5

(a-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:46,(d-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-5) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 6

(a-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:49,(d-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-6) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-6) a CDR comprising the amino acid sequence shown in SEQ ID

-   -   35 NO

Group 7

(a-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:52,(d-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-7) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 8

(a-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:43,(d-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-8) a CDR comprising the amino acid sequence shown in SEQ ID NO:55

Group 9

(a-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:60,(b-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:5, and(f-9) a CDR comprising the amino acid sequence shown in SEQ ID NO:61

Group 10

(a-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:66,(d-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-10) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 11

(a-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:69,and(f-11) a CDR comprising the amino acid sequence shown in SEQ ID NO:70

Group 12

(a-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:60,(b-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:66,(d-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-12) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 13

(a-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:77,(e-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-13) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 14

(a-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:80,(b-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:81,and(f-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 15

(a-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-15) a CDR comprising the amino acid sequence shown in SEQ ID NO: 66,(d-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-15) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-15) a CDR comprising the amino acid sequence shown in SEQ ID NO: 6

Group 16

(a-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:90,(d-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-16) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 17

(a-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-17) a CDR comprising the amino acid sequence shown in SEQ ID NO: 52,(d-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-17) a CDR comprising the amino acid sequence shown in SEQ ID NO:93

Group 18

(a-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:98,(b-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-18) a CDR comprising the amino acid sequence shown in SEQ ID NO:99

Group 19

(a-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:60,(b-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-19) a CDR comprising the amino acid sequence shown in SEQ ID NO: 3,(d-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-19) a CDR comprising the amino acid sequence shown in SEQ ID NO:99

Group 20

(a-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:90,(d-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 21

(a-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-21) a CDR comprising the amino acid sequence shown in SEQ ID NO:55

Group 22

(a-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:66,(d-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:110,(e-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-22) a CDR comprising the amino acid sequence shown in SEQ ID NO:6

Group 23

(a-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and(f-23) a CDR comprising the amino acid sequence shown in SEQ ID NO:115

Group 24

(a-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:90,(d-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:120,and(f-24) a CDR comprising the amino acid sequence shown in SEQ ID NO:6; or(2) an antibody comprising CDRs of (a-X) to (f-X) (X is 1 to 24; thesame applies below) above, wherein one or more, preferably one toseveral (e.g., 1, 2, 3, 4 or 5) amino acid residues are substitutedand/or deleted and/or added and/or inserted in one or more (e.g., 1, 2,3, 4, 5 or 6) amino acid sequences selected from the amino acidsequences shown in (a-X) to (f-X), and wherein the antibody has abinding property equivalent to that of any one of the above-mentionedantibodies, or binds to wild type human CD81 competitively with any oneof the above-mentioned antibodies. Namely, the “equivalent bindingproperty” means at least binding to a peptide region consisting of the80th to 175th or 113th to 175th amino acid residues of wild type humanCD81 polypeptide, preferably further binding to at least one human CD81variant selected from (1) to (13) mentioned above (more preferably thevariants (9) and (11)) in a binding affinity of less than 40% when itsbinding affinity to wild type human CD81 is 100%.

The antibody (2) above can be obtained by a publicly known method, forexample, by performing PCR with a vector that encodes the base sequenceof the variable region of the antibody (1) above as the template tocomprehensively introduce mutations to generate a library of phagedisplay mutant antibodies, screening the library with the bindingactivity for human CD81 or the competitive binding against the antibody(1) above as an index, and performing panning.

The binding property and competitive binding of an antibody can bedetermined by various binding assays and competitive assays describedabove. As a result of the assays, when the “equivalent binding property”or “competitive binding” of the tested antibody is confirmed, then itstherapeutic effect can be tested by the cell migration experimentdescribed in detail below.

A more preferable example of the antibodies having the bindingproperties mentioned above is an antibody comprising:

(1) a light chain variable region comprising the above-identified CDRs(a-X) to (c-X) and a heavy chain variable region comprising theabove-identified CDRs (d-X) to (f-X); or(2) the light chain and heavy chain variable region of (1) above,wherein one or more, preferably one to several (e.g., 1, 2, 3, 4 or 5)amino acid residues are substituted and/or deleted and/or added and/orinserted in one or more (e.g., 1, 2, 3, 4, 5 or 6) amino acid sequencesselected from the amino acid sequences shown in (a-X) to (f-X), andwherein the antibody has a binding property equivalent to that of anyone of the above-mentioned antibodies, or binds to wild type human CD81competitively with any one of the above-mentioned antibodies. Here, the“equivalent binding property” means the same as above.

More preferably, in the antibody mentioned above, the CDRs (a-X), (b-X)and (c-X) are located in this order from the N-terminus of the lightchain. Namely, the CDRs (a-X), (b-X) and (c-X) corresponds to CDR1, CDR2and CDR3 of the light chain, respectively. Similarly, the CDRs (d-X),(e-X) and (f-X) are located in this order from the N-terminus of theheavy chain. Namely, the CDRs (d-X), (e-X) and (f-X) correspond to CDR1,CDR2 and CDR3 of the heavy chain, respectively.

An even more preferable example of the antibodies having the bindingproperties mentioned above is an antibody comprising:

(1) a light chain variable region comprising the amino acid sequenceshown in (i-X) and a heavy chain variable region comprising the aminoacid sequence shown in (j-X); or(2) the light chain and heavy chain variable region of (1) above,wherein one or more, preferably 1 to 20, more preferably 1 to 10, evenmore preferably one to several (e.g., 1, 2, 3, 4 or 5) amino acidresidues are substituted and/or deleted and/or added and/or inserted ineither or both of the amino acid sequences shown in (i-X) and (j-X), andwherein the antibody has a binding property equivalent to that of anyone of the above-mentioned antibodies, or binds to wild type human CD81competitively with any one of the above-mentioned antibodies. Here, the“equivalent binding property” means the same as above.

Another preferable example of the antibodies having the bindingproperties mentioned above is an antibody that binds to the same oressentially the same epitope of wild type human CD81 as that to whichthe antibody comprising a light chain variable region comprising theamino acid sequence shown in (i-X) and a heavy chain variable regioncomprising the amino acid sequence shown in (j-X) binds. A morepreferable example is an antibody that binds to the same or essentiallythe same epitope of wild type human CD81 as that to which the antibodycomprising a light chain comprising the amino acid sequence shown in(k-X) and a heavy chain comprising the amino acid sequence shown in(l-X) binds.

Here, “essentially the same epitope” means an epitope that is differentfrom, but sterically overlaps, the epitope recognized by an antibodyhaving the above-mentioned particular light chain (variable region) andheavy chain (variable region) sequences. An antibody recognizing“essentially the same epitope” competes with an antibody having theabove-mentioned particular light chain (variable region) and heavy chain(variable region) sequences for binding to wild type human CD81.

The most widely used and rapid methods for determining whether twoantibodies bind to identical or sterically overlapping epitopes arecompetition assays, which can be configured in all number of differentformats, using either labeled antigen or labeled antibody. Usually, theantigen is immobilized on a substrate, and the ability of unlabeledantibodies to block the binding of labeled antibodies is measured usingradioactive isotopes or enzyme labels.

The antibody of the present invention may also be an antibody comprisingat least one CDR selected from among six CDRs belonging to any one ofthe groups 1 to 24 above, that binds to wild type human CD81competitively with an antibody comprising all the six CDRs.

Furthermore, the antibody of the present invention may be an antibodythat possesses a homology of 80% or more, preferably 90% or more, morepreferably 95% or more, to the amino acid sequences of the light chainand heavy chain of an antibody comprising all six CDRs belonging to anyone of the groups 1 to 24 above, and binds to wild type human CD81competitively with the antibody. Amino acid sequence homology asmentioned herein can be calculated using the blastp program of NCBIBLAST (National Center for Biotechnology Information Basic LocalAlignment Search Tool) under the following conditions (expectancy-10;gap allowed; matrix-BLOSUM62; filtering-OFF).

(ii) Another Specific Embodiment

Another preferable embodiment is an antibody having a binding affinityof less than 40% to each of the human CD81 variants described in (1) to(5), (7), (8), (11) and (12) when its binding affinity to wild typehuman CD81 is 100%.

A preferable example of the antibodies having the binding propertiesmentioned above

(1) an antibody comprising:(a-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:11;(b-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:12;(c-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:13;(d-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:14;(e-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:15;and(f-25) a CDR comprising the amino acid sequence shown in SEQ ID NO:16,or(2) an antibody comprising CDRs of (a-25) to (f-25) above,wherein one or more, preferably one to several (e.g., 1, 2, 3, 4 or 5)amino acid residues are substituted and/or deleted and/or added and/orinserted in one or more (e.g., 1, 2, 3, 4, 5 or 6) amino acid sequencesselected from the amino acid sequences shown in SEQ ID NOs:11 to 16, andwherein the antibody has a binding property equivalent to that of anyone of the above-mentioned antibodies, or binds to wild type human CD81competitively with any one of the above-mentioned antibodies. Here, the“equivalent binding property” means the same as above.

The binding property and competitive binding of an antibody can bedetermined by various binding assays and competitive assays describedabove. As a result of the assays, when the “equivalent binding property”or “competitive binding” of the tested antibody is confirmed, then itstherapeutic effect can be tested by the cell migration experimentdescribed in detail below.

A more preferable example of the antibodies having the bindingproperties mentioned above is an antibody comprising:

(1) a light chain variable region comprising the above-identified CDRs(a-25) to (c-25) and a heavy chain variable region comprising theabove-identified CDRs (d-25) to (f-25); or(2) the light chain and heavy chain variable region of (1) above,wherein one or more, preferably one to several (e.g., 1, 2, 3, 4 or 5)amino acid residues are substituted and/or deleted and/or added and/orinserted in one or more (e.g., 1, 2, 3, 4, 5 or 6) amino acid sequencesselected from the amino acid sequences shown in SEQ ID NOs:11 to 16, andwherein the antibody has a binding property equivalent to that of anyone of the above-mentioned antibodies, or binds to wild type human CD81competitively with any one of the above-mentioned antibodies. Here, the“equivalent binding property” means the same as above.

More preferably, in the antibody mentioned above, the CDRs (a-25),(b-25) and (c-25) are located in this order from the N-terminus of thelight chain. Namely, the CDRs (a-25), (b-25) and (c-25) corresponds toCDR1, CDR2 and CDR3 of the light chain, respectively. Similarly, theCDRs (d-25), (e-25) and (f-25) are located in this order from theN-terminus of the heavy chain. Namely, the CDRs (d-25), (e-25) and(f-25) correspond to CDR1, CDR2 and CDR3 of the heavy chain,respectively.

An even more preferable example of the antibodies having the bindingproperties mentioned above is an antibody comprising:

(1) a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO:18 and a heavy chain variable region comprising theamino acid sequence shown in SEQ ID NO:20; or(2) the light chain and heavy chain variable region of (1) above,wherein one or more, preferably 1 to 20, more preferably 1 to 10, evenmore preferably one to several (e.g., 1, 2, 3, 4 or 5) amino acidresidues are substituted and/or deleted and/or added and/or inserted ineither or both of the amino acid sequences shown in SEQ ID NOs:18 and20, and wherein the antibody has a binding property equivalent to thatof any one of the above-mentioned antibodies, or binds to wild typehuman CD81 competitively with any one of the above-mentioned antibodies.Here, the “equivalent binding property” means the same as above.

Another preferable example of the antibodies having the bindingproperties mentioned above is an antibody that binds to the same oressentially the same epitope of wild type human CD81 as that to whichthe antibody comprising a light chain variable region comprising theamino acid sequence shown in SEQ ID NO:18 and a heavy chain variableregion comprising the amino acid sequence shown in SEQ ID NO:20 binds. Amore preferable example is an antibody that binds to the same oressentially the same epitope of wild type human CD81 as that to whichthe antibody comprising a light chain comprising the amino acid sequenceshown in SEQ ID NO:30 and a heavy chain comprising the amino acidsequence shown in SEQ ID NO:32 binds. Here, “essentially the sameepitope” mean the same as above.

The antibody of the present invention may also be an antibody thatcomprises at least one CDR selected from among the six CDRs (a-25) to(f-25) above, and binds to wild type human CD81 competitively with anantibody comprising all the six CDRs.

Furthermore, the antibody of the present invention may be an antibodythat possesses a homology of 80% or more, preferably 90% or more, morepreferably 95% or more, to the amino acid sequences of the light chainand heavy chain of an antibody comprising all the six CDRs (a-25) to(f-25) above, and binds to wild type human CD81 competitively with theantibody. Here, amino acid homology can be calculated in the same manneras the above.

(I) Production of Antibody

The antibody of the present invention can be any monoclonal antibody, aslong as it binds to the particular peptide region of the wild type humanCD81 mentioned above. Although the isotype of the antibody is notsubject to limitation, it is preferably IgG, IgM or IgA, particularlypreferably IgG. Also, the molecule type of the antibody is not subjectto limitation, in addition to the entire antibody molecule, the antibodymay, for example, be a fragment such as Fab, Fab′, or F(ab′)₂, agenetically engineered conjugate molecule such as scFv, scFv-Fc,minibody, or diabody, or a derivative thereof modified with a certainmolecule, for example, a molecule having a stabilizing action such aspolyethylene glycol (PEG), and the like.

Since the antibody of the present invention is used as a pharmaceuticalproduct having humans as the subject of administration thereof, theantibody used in the present invention is an antibody whose risk ofshowing antigenicity when administered to a human has been reduced; tobe specific, the antibody is a (fully) human antibody, a humanizedantibody, a non-human (e.g., mouse, rat, rabbit)-human chimeric antibodyand the like, particularly preferably a human antibody. A humanizedantibody and a chimeric antibody can be prepared by genetic engineeringtechnology according to the method described below. Although a (fully)human antibody can also be produced from human-human (or human-mouse)hybridoma, it is desirable to produce it using a phage display method ora human antibody-producing animal as described below (e.g., mouse), inorder to stably supply the antibody in large amounts at low costs.

(II) Production Method of the Antibody of the Present Invention

The antibody of the present invention can be produced by the methoddescribed in the following Examples or a known method.

(i) Preparation of Antigen

The antigen used to prepare the antibody of the present invention may bethe wild type human 0081 protein or partial peptide thereof, a(synthetic) peptide having one or more kinds of the same antigendeterminant as that thereof and the like (hereinafter these aresometimes simply referred to as the antigen of the present invention).

The wild type human CD81 protein or a partial peptide thereof isproduced by, for example, (a) preparing the same from a human tissue orcells, by a method known to the public or its modified method (b)chemically synthesizing the same by a publicly known method of peptidesynthesis using a peptide synthesizer and the like, (c) culturing atransformant comprising a DNA that encodes wild type human CD81 or apartial peptide thereof, or (d) biochemically synthesizing the same witha nucleic acid that encodes wild type human CD81 or a partial peptidethereof as the template using a cell-free transcription/translationsystem.

(iia) Preparation of Human Antibody Using Phage Display Human AntibodyLibrary

A human antibody can be produced by phage display (Hoogenboom andWinter, J. Mod. Biol., 227:381 (1991); Marks et al, J. Mol. Biol.,222:581 (1991); Cole et al, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, p. 77 (1985) and Boermer et al, J. Immunol., 147(1):86-95(1991)).

The method of preparing a phage display human antibody library include,but are not limited to, for example, the methods described below.

Although a phage used is not subject to limitation, filamentous phage(Ff bacteriophage) is normally preferably used. As the method ofpresenting a foreign protein on the phage surface, a method comprisingexpressing and presenting the foreign protein as a fusion protein withany of the coat proteins g3p, and g6p to g9p on the coat protein can bementioned; and a method comprising fusing the foreign protein to theN-terminal side of g3p or g8p is often used. As the phage displayvector, besides 1) one in which the foreign gene is introduced in theform of fusion gene with the coat protein gene of the phage genome, toallow all the coat proteins presented on the phage surface to bepresented as a fusion protein with the foreign protein, 2) one in whichthe gene encoding the fusion protein is inserted separately from thewild-type coat protein gene to allow the fusion protein and thewild-type coat protein to be expressed simultaneously, and 3) an E. colihaving a phagemid vector harboring the gene that encodes the fusionprotein is infected with a helper phage having the wild-type coatprotein gene to produce phage particles that express the fusion proteinand the wild-type coat protein simultaneously, and the like can bementioned. However, a phage display vector of the type 2) or 3) is usedfor the preparation of an antibody library, because in the case of 1),the capability of infection is lost when a large foreign protein isfused.

As a specific vector, those described by Holt et al. (Curr. Opin.Biotechnol., 11: 445-449, 2000) can be mentioned as examples. Forexample, pCES1 (see J. Biol. Chem., 274: 18218-18230, 1999) is anFab-expressing phagemid vector wherein a DNA encoding the κL chainconstant region allocated to downstream of the g3p signal peptide, and aDNA encoding CH3, His-tag, c-myc tag, and the amber stop codon (TAG)followed by the g3p coding sequence, allocated to downstream of the g3psignal peptide, are arranged under the control of one lactose promoter.When this is introduced to an E. coli having an amber mutation, Fab ispresented onto the g3p coat protein, but when it is expressed in theHB2151 strain and the like, which do not have an amber mutation, asoluble Fab antibody is produced. As the scFv-expressing phagemidvector, for example, pHEN1 (J. Mol. Biol., 222: 581-597, 1991) and thelike are used.

Meanwhile as examples of the helper phage, M13-KO7, VCSM13 and the likecan be mentioned.

And as another phage display vector, a vector that is designed as a DNAsequence comprising the cysteine-encoding codon is linked to each of the3′ end of the antibody gene and the 5′ end of the coat protein gene toexpress the two genes simultaneously and separately (not in the form ofa fusion protein), and to present the antibody onto the coat protein onthe phage surface via S—S bonds between the introduced cysteine residues(CysDisplay™ technology of Morphosys Company) and the like, can bementioned.

As the kind of human antibody library, a naive/non-immunized library, asynthetic library, an immunized library and the like can be mentioned.

The naive/non-immunized library is a library obtained by acquiring theV_(H) and V_(L) genes retained by a normal human by RT-PCR, and randomlycloning them into the above-described phage display vector. Normally,mRNA derived from lymphocytes of peripheral blood, bone marrow, tonsiland the like of a normal human, and the like are used as the template. Alibrary prepared by selectively amplifying IgM-derived mRNA in which aclass switch due to antigen sensitization is not undergoing, to avoid Vgene biases such as clinical history, is particularly called a naivelibrary. Representatively, the library of Cambridge Antibody Technology(see J. Mol. Biol., 222: 581-597, 1991; Nat. Biotechnol., 14: 309-314,1996), the library of Medical Research Council (see Annu. Rev. Immunol.,12: 433-455, 1994), the library of Dyax Corp. (see J. Biol. Chem., 1999(supra); Proc. Natl. Acad. Sci. USA, 14: 7969-7974, 2000) and the likecan be mentioned.

A synthetic library is obtained by selecting a functional particularantibody gene in human B cells, and substituting a portion ofantigen-binding region in a V gene segment, for example, CDR3 and thelike, with DNAs encoding a random amino acid sequence of appropriatelength, to construct a library. It is recognized to be excellent inantibody expression efficiency and stability because the library can beconstructed with the combination of the V and V_(L) genes, which producefunctional scFv and Fab, since the beginning. Representatively, theHuCAL library of Morphosys AG (see J. Mol. Biol., 296: 57-86, 2000), thelibrary of Bioinvent (see Nat. Biotechnol., 18: 852, 2000), the libraryof Crucell (see Proc. Natl. Acad. Sci. USA, 92: 3938, 1995; J. Immunol.Methods, 272: 219-233, 2003) and the like can be mentioned.

An immunized library is a library obtained by preparing an mRNA fromlymphocytes collected from a human such as a patient with cancer,autoimmune disease, infectious disease and the like or a recipient ofvaccination, having an elevated blood antibody titer against the targetantigen, or from human lymphocytes and the like which are artificiallyimmunized with the target antigen by the above-described in vitroimmunization method, in the same manner as with the above-describednaive/non-immunized library, and amplifying the V_(H) and V_(L) genes byRT-PCR, to construct a library. It is possible to obtain the desiredantibody even from such libraries of relatively small size because thedesired antibody gene is contained in the library already at thebeginning.

The process for selecting an antibody against the target antigen by thephage display method is referred to as panning. To be specific, forexample, a phage presenting an antigen-ic) specific antibody isconcentrated by repeating a series of operations of bringing anantigen-immobilized carrier and a phage library into contact with eachother, washing out the unbound phage, thereafter eluting the bound phagefrom the carrier, and infecting the phage to E. coli to proliferate it,about 3 to 5 times. As the carrier for immobilizing the antigen, variouscarriers used in ordinary antigen-antibody reactions or affinitychromatography, for example, insoluble polysaccharides such as agarose,dextran, and cellulose, synthetic resins such as polystyrene,polyacrylamide, and silicon, or microplates, tubes, membranes, columns,beads and the like comprising glass, metal and the like, and surfaceplasmon resonance (SPR) sensor chips, and the like can be mentioned. Forthe antigen immobilization, physical adsorption may be used, and amethod using a chemical bond used to insolubilize and immobilize aprotein or enzyme and the like is also acceptable. For example, abiotin-(strept)avidin system and the like are preferably used. Forwashing the unbound phage, a blocking solution such as BSA solution(once or twice), a PBS comprising a surfactant such as Tween (3 to 5times) and the like can be used. There is also a report mentioning thatthe use of citrate buffer (pH 5) and the like is preferable for thewashing. For elution of the specific phage, an acid (e.g., 0.1 Mhydrogen chloride and the like) is normally used; cleavage with aspecific protease (e.g., a gene sequence that encodes the trypsincleavage site can be introduced into the linkage site between theantibody gene and the coat protein gene. In this case, E. coli infectionand proliferation are possible even if all the coat protein is expressedin the form of a fusion protein because the wild-type coat protein ispresented on the surface of the eluted phage), competitive elution witha soluble antigen, or elution by reduction of S—S bond (e.g., in theaforementioned CysDisplay™ the antigen-specific phage can be collectedby dissociating the antibody and the coat protein by using a suitablereducing agent after performing panning) is also possible. When elutionhas been performed with an acid, the eluate is neutralized with Tris andthe like, and the eluted phage is then infected to E. coli, which iscultured; after which the phage is collected by a conventional method.

After the phage presenting the antigen-specific antibody is concentratedby panning, the phage is infected to E. coli and the cells are sown ontoa plate to perform cell cloning. The phage is again collected from theeach clone, and the antigen binding activity is confirmed by theabove-described antibody titer assay (e.g., ELISA, RTA and the like) ora measurement utilizing FACS or SPR.

Isolation and purification of the antibody from the selected phage clonethat presents the antigen-specific antibody can be performed by, forexample, when using a vector incorporating an amber stop codon at thelinker site of the antibody gene and the coat protein gene as the phagedisplay vector, infecting the phage to an E. coli that does not haveamber mutation (e.g., HB2151 strain) to produce and secrete solubleantibody molecules in periplasm or the medium, lysing the cell wall withlysozyme and the like, collecting the extracellular fraction, andpurifying using the same purification technique as described above.Provided that the His-tag or c-myc tag has been introduced in advance,the antibody can easily be purified by using Immobilized Metal AffinityChromatography (IMAC) method, an anti-c-myc antibody column and thelike. When cleavage with a specific protease is utilized in panning, theantibody molecule is separated from the phage surface by an action withthe protease, so that the desired antibody can be purified by performingthe same purification operation as above mentioned.

The affinity and avidity of the human antibody (e.g., scFv, Fab) thusobtained to the epitope region of wild type human CD81 is confirmed bythe binding assays mentioned above using a polypeptide comprising thepeptide region.

(iib) Preparation of Human Antibody Using Human Antibody-ProducingAnimal

Provided that a functional human Ig gene is introduced into a non-humanwarm-blooded animal having the endogenous immunoglobulin (Ig) geneknocked out (KO) therein, and that this animal is immunized with anantigen, a human antibody is produced in place of the antibody derivedfrom the animal. Therefore, provided that an animal such as mice, forwhich a technique for producing a hybridoma has been established, isused, it is possible to acquire a fully human monoclonal antibody by thesame method as the conventional method used to prepare a mousemonoclonal antibody. Namely, human monoclonal antibodies can begenerated by using a human antibody-producing mouse (see Immunol. Today,17: 391-397, 1996) obtained by crossing a mouse transfected withminigenes of the human Ig H chain and L chain using an ordinarytransgenic (Tg) technique with a mouse wherein the endogenous mouse Iggene has been inactivated using an ordinary KO technique (WO 93/12227,WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

The hybridoma obtained by fusion of the anti-human CD81antibody-producing cell obtained from the human antibody-producing mousewith a myeloma cell are cultured, and the anti-human CD81 antibody canbe recovered from the culture supernatant and purified by conventionalmethods. The affinity and avidity of the human antibody thus obtained tothe epitope region of wild type human CD81 is confirmed by the bindingassays mentioned above using a polypeptide comprising the peptideregion.

(iic) Preparation of Chimeric Antibody

As used herein, “chimeric antibody” means an antibody wherein thesequences of the variable regions of the H chain and L chain (V_(H) andV_(L)) thereof are derived from a non-human animal species, and whereinthe sequences of the constant regions (C_(H) and C_(L)) are derived fromhuman. The sequences of the variable regions are preferably derivedfrom, for example, an animal species permitting easy preparation of ahybridoma, such as mouse, rat, rabbit and the like.

As examples of the method of preparing a chimeric antibody, the methoddescribed in U.S. Pat. No. 6,331,415 or a partially modified methodthereof and the like can be mentioned.

Host cells are transformed with the chimeric H chain and chimeric Lchain expression vector(s) obtained. As the host cells, animal cells,for example, Chinese hamster ovary (CHO) cells, monkey-derived COS-7cells, Vero cells, rat-derived GHS cells and the like, in addition tothe above-described mouse myeloma cells, can be mentioned. For thetransformation, any method applicable to animal cells can be used, withpreference given to electroporation method and the like. It is possibleto isolate a chimeric monoclonal antibody by culturing the host cells ina medium suitable thereto for a given period, and thereafter collectingthe culture supernatant and purifying it in the same manner as describedabove. Alternatively, it is also possible to obtain a chimericmonoclonal antibody easily and in large amounts from milk or eggs oftransgenic animals which are produced by a conventional method usinggerm line cells of an animal such as bovine, goat, or fowl as the hostcells, for which a transgenic technique has been established and aknow-how of mass propagation as a domestic animal (domestic fowl) hasbeen compiled. Furthermore, it is also possible to obtain a chimericmonoclonal antibody in large amounts from the seeds, leaves and the likeof a transgenic plant, produced by using microinjection andelectroporation into protoplast, the particle gun method and Ti-vectormethod for intact cells and the like, with cells of a plant such ascorn, rice, wheat, soybean, or tobacco as the host cells, for which atransgenic technique has been established, and which is cultured inlarge amounts as a major crop.

(iii) Humanized Antibody

As used herein, “a humanized antibody” means an antibody wherein thesequences of all regions present in the variable region, other than thecomplementarity determining region (CDR), [i.e., framework region (FR)in constant region and variable region] are derived from a human, andwherein only the sequence of CDR is derived from another mammalianspecies. The other mammalian species is preferably an animal species,for example, mouse, rat, rabbit and the like, with which production ofhybridomas can be easily performed.

As examples of the method of preparing a humanized antibody, the methodsdescribed in U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761 and5,693,762, EP 239400, WO 92/19759 or partially modified methodstherefrom and the like can be mentioned. To be specific, DNAs thatencode V_(H) and V_(L) derived from a non-human mammalian species (e.g.,mouse) are isolated in the same manner as with the above-describedchimeric antibody, after which sequencing is performed by a conventionalmethod using an automated DNA sequencer (e.g., manufactured by AppliedBiosystems Company and the like), and the nucleotide sequences obtainedor deduced amino acid sequences therefrom are analyzed using a knownantibody sequence database [for example, Kabat database (see Kabat etal., “Sequences of Proteins of Immunological Interest”, edited by NIH,US Department of Health and Human Services, Public Health Service, 5thedition, 1991) and the like] to determine the CDR and FR of the twochains. A nucleotide sequence wherein the CDR encoding region of anucleotide sequence that encodes the L chain and H chain of a humanantibody having an FR sequence similar to the determined FR sequence issubstituted with the determined nucleotide sequence that encodes the CDRof another animal species, is designed, and the nucleotide sequence isdivided into fragments of about 20 to 40 bases, and a sequencecomplementary to the nucleotide sequence is divided into fragments ofabout 20 to 40 bases so that they alternatively overlap with theaforementioned fragments. It is possible to construct DNAs that encodeV_(H) and V_(L) having human-derived FR and a CDR derived from anothermammalian species by synthesizing individual fragments using a DNAsynthesizer, and hybridizing and ligating them in accordance withconventional methods. In order to transfer a CDR derived from anothermammalian species into human-derived V_(H) and V_(L) more quickly andmore efficiently, it is preferable to use PCR-based site directedmutagenesis. As examples of such a method, the sequential CDR graftingmethod described in JP-A-5-227970 and the like can be mentioned.

It should be noted that in preparing a humanized antibody by a method asdescribed above, the antigen binding activity may sometimes decrease,compared with the original non-human antibody, if the amino acidsequence of CDR alone is transplanted to the template human antibody FR.In such cases, it is effective to transplant some FR amino acids aroundthe CDR in combination. The non-human antibody FR amino acid to betransplanted may be an amino acid residue that is important to themaintenance of the steric structure of each CDR; such an amino acidresidue can be deduced by a steric structure estimation using acomputer.

It is possible to obtain cells or transgenic animal/plant that producesa humanized antibody by ligating the thus-obtained DNAs encoding V_(H)and V_(L) to DNAs encoding human-derived C_(H) and C_(L), respectively,and introducing the ligated product into suitable host cells.

An alternative method of preparing a humanized antibody without usingCDR grafting wherein mouse CDRs are grafted into variable regions of ahuman antibody is, for example, a method comprising determining which isan amino acid residue that can be substituted in a non-human variableregion, on the basis of a conserved structure-function correlationbetween antibodies. This method can be carried out as described in, forexample, EP 0571613 B1, U.S. Pat. No. 5,766,886, U.S. Pat. No.5,770,196, U.S. Pat. No. 5,821,123, U.S. Pat. No. 5,869,619 and thelike. Provided that the amino acid sequence information on each of theV_(H) and V_(L) of the original non-human antibody, preparation of ahumanized antibody using the method can easily be performed byutilizing, for example, the contract antibody preparation serviceprovided by Xoma.

A humanized antibody, like a chimeric antibody, can be modified to scFv,scFv-Fc, minibody, dsFv, Fv and the like by using genetic engineeringtechniques; and they can be produced in a microorganism such as E. colior yeast by using a suitable promoter.

The affinity and avidity of the humanized antibody thus obtained to theepitope region of wild type human CD81 is confirmed by the bindingassays mentioned above using a polypeptide comprising the peptideregion.

(III) Optimization of Antibody

The anti-human CD81 antibodies of the invention mentioned above may bereadily prepared to include various changes, substitutions, insertions,and deletions. For example, to maximize the expression level of anantibody, the nucleotide sequences of the antibody gene may be optimizedso as to match the codon usage frequency of the cell used for antibodyexpression. Additional modifications to enhance antibody stabilityinclude modification of IgG4 to replace the serine at residue 228 in thehinge region with proline as described below. As other modifications,substitutions as required to optimize efficiency in conjugating theantibody with a drug can be mentioned. For example, an antibody may bemodified at its carboxyl terminus to include amino acids for drugattachment, for example, one or more cysteine residues may be added. Theconstant regions may be modified to introduce sites for binding ofcarbohydrates or other moieties.

Mutants of anti-CD81 antibodies of the invention may be produced usingstandard recombinant techniques, including site-directed mutagenesis, orrecombination cloning.

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable regions are mutated to generate all possible aminosubstitutions at each site. The antibody variants thus generated aredisplayed as fused proteins with a coat protein g3p of filamentous phageM13 on phages. The phage-displayed variants are then screened for theirbiological activity (e.g., binding affinity) as herein disclosed. Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development. Nucleicacid molecules encoding amino acid sequence variants of the anti-humanCD81 antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-human CD81antibody.

Preferred affinity matured antibodies have an affinity which is fivetimes, more preferably 10 times, even more preferably 20 or 30 timesgreater than the starting antibody from which the matured antibody isprepared.

IgG4 Mutation

When an IgG4 antibody is administered to human or some animals, aphenomenon that the serum concentration of IgG4 antibody is reduced dueto swapping with an endogenous IgG4 has been observed. It has been knownthat this IgG4 exchange is inhibited by the point mutation of Ser atresidue 228 with Pro (Mol. Immunol. 30, 105, 1993, Protein Sci. 6, 407,1997, Mol. Immunol. 38, 1, 2001, Nat. Biotechnol. 27, 767, 2009).

(IV) Confirmation of Therapeutic Effect of Antibody

The antibody of the present invention has a suppressive effect on Tlymphocyte migration. This action is detected using a widely knownevaluation system for lymphocyte function. For example, the effect on Tlymphocyte migration is evaluated by bringing human peripheral bloodmononuclear cells (PBMCs) cultured in the presence of cytokines (e.g.,PHA, IL-2, TNF-α, IL-22, IL-7 and VIP) into contact with a testantibody, stimulating the migration of PBMCs (e.g., by adding achemokine, stromal cell-derived factor 1 (SDF-1; also known as CXCL12),and determining inhibition of the migration (see, for example, Blood2009 August 13; 114(7): 1366-1373). To be specific, a cell migrationexperiment described below can be used.

As the lymphocytes, established cell lines such as Jurkat cells (CloneE6-1, ATCC Number TIB-152), and cells derived from human peripheralblood can be used. Human peripheral blood-derived cells are commerciallyavailable (KAC Co., Ltd. and the like). Alternatively, human peripheralblood-derived cells can also be prepared by isolating a mononuclear cellfraction containing monocytes and lymphocytes from a peripheral blood ofa healthy human using Ficoll/Paque density gradient centrifugationaccording to The Journal of Immunology, 147, 2251 (1991).

Cell Migration Experiment (Chemotaxis Assay)

The ability of an antibody or a functional fragment thereof to inhibitits antigen's function relating to cell migration can be evaluated usinga cell migration experiment (chemotaxis assay). In general, a cellmigration experiment is performed by separating appropriate cells suchas leukocytes (e.g., lymphocytes, eosinophils, basophils) and achemotactic factor by a barrier (e.g., endothelium, filter), andmonitoring the migration of the cells toward or across the barrier. Forexample, the suppressive effect on cell migration. of a test antibodycan be examined by adding a culture medium containing a chemotacticfactor such as SDF-1 to a well of 96-well migration plate (the firstchamber), putting a transwell having the bottom surface consisting of acell-permeable macroporous membrane (the second chamber) onto the well,adding the test antibody and the cells, measuring the migration of thecells from the second chamber to the first chamber and comparing theextent of migration with that in the absence of antibody or in thepresence of a non-specific antibody (Immunol. Invest. 17: 625-677(1988)).

(V) Production of Recombinant Antibody

Any production system can be used for the production of the antibodyused in the present invention. The production systems for producingantibodies include in vitro and in vivo production systems. In vitroproduction systems include production systems using eukaryotic cells andthose using prokaryotic cells.

Antibody genes constructed as described above may be expressed andobtained in a known method. In the case of mammalian cells, expressionmay be accomplished using a DNA in which a commonly used usefulpromoter, the antibody gene to be expressed, and the poly A signal at 3′downstream thereof have been functionally linked or a vector containingsaid DNA. Examples of the promoter/enhancer include humancytomegalovirus immediate early promoter/enhancer.

Additionally, as the promoter/enhancer which can be used for expressionof antibody for use in the present invention, there are viralpromoters/enhancers such as retrovirus, polyoma virus, adenovirus, andsimian virus 40 (SV40), and promoters/enhancers derived from mammaliancells such as human elongation factor 1α. (hEF1α).

For example, expression may be readily accomplished by the method ofMulligan et al. (Nature (1979) 277, 108) when SV40 promoter/enhancer isused, or by the method of Mizushima et al. (Nucleic Acids Res. (1990)18, 5322) when hEF1α. promoter/enhancer is used.

In the case of E. coli, expression may be conducted by functionallylinking a commonly used useful promoter, a signal sequence for antibodysecretion, and the antibody gene to be expressed, followed by expressionthereof. As the promoter, for example, there can be mentioned lacZpromoter and araB promoter. The method of Ward et al. (Nature (1989)341, 544-546; FASEB J. (1992) 6, 2422-2427) may be used when lacZpromoter is used, and the method of Better et al. (Science (1988) 240,1041-1043) may be used when araB promoter is used.

As the signal sequence for antibody secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379) can be used. After separating the antibodyproduced in the periplasm, the structure of the antibody isappropriately refolded before use (see, for example, WO 96/30394).

As the origin of replication, there can be used those derived from SV40,polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like.Furthermore, for the amplification of the gene copy number in the hostcell system, expression vectors can include as selectable markers theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene and the like.

When the eukaryotic cells are used, there are the production systemswhich employ animal cells, plant cells, and fungal cells. Known animalcells include (1) mammalian cells such as CHO cells, COS cells, myelomacells, baby hamster kidney (BHK) cells, HeLa cells, and Vero cells, (2)amphibian cells such as Xenopusoocytes, or (3) insect cells such as sf9,sf21, and Tn5. Known plant cells include, for example, those derivedfrom Nicotiana tabacum, which is subjected to callus culture. Knownfungal cells include yeasts such as the genus Saccharomyces, morespecifically Saccharomyces cerevisiae, or filamentous fungi such as thegenus Aspergillus, more specifically Aspergillus niger.

When the prokaryotic cells are used, there are the production systemswhich employ bacterial cells. Known bacterial cells include Escherichiacoli (E. coli), and Bacillus subtilis.

By introducing via transformation the gene of the desired antibody intothese cells and culturing the transformed cells in vitro, the antibodycan be obtained. Culturing is conducted in the known methods. Forexample, as the culture medium DMEM, MEM, RPMI1640, and IMDM can beused, and serum supplements such as fetal calf serum (FCS) may be usedin combination. In addition, antibodies may be produced in vivo byimplanting cells into which the antibody gene has been introduced intothe abdominal cavity of an animal and the like.

As in vivo production systems, there can be mentioned those which employanimals and those which employ plants. When animals are used, there arethe production systems which employ mammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle can be used (VickiGlaser, SPECTRUM Biotechnology Applications, 1993). Also, as insects,silkworms can be used.

When plants are used, tabacco, for example, can be used.

Antibody genes are introduced into these animals or plants, and theantibodies are produced in such animals or plants, and recovered. Forexample, an antibody gene is inserted into the middle of the geneencoding protein which is inherently produced in the milk such as goatβ-casein to prepare fusion genes. DNA fragments containing the fusiongene into which the antibody gene has been inserted are injected into agoat embryo, and the embryo is introduced into a female goat. Thedesired antibody is obtained from the milk produced by the transgenicgoat born to the goat who received the embryo or off springs thereof. Inorder to increase the amount of milk containing the desired antibodyproduced by the transgenic goat, hormones may be given to the transgenicgoat as appropriate. (Ebert, K. M. et al., Bio/Technology (1994) 12,699-702).

When silkworms are used, baculovirus into which the desired antibodygene has been inserted is infected to the silkworm, and the desiredantibody can be obtained from the body fluid of the silkworm (Maeda, S.et al., Nature (1985) 315, 592-594). Moreover, when tabacco is used, thedesired antibody gene is inserted into an expression vector for plants,for example pMON 530, and then the vector is introduced into a bacteriumsuch as Agrobacterium tumefaciens. The bacterium is then infected totabacco such as Nicotiana tabacum to obtain the desired antibody fromthe leaves of the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol.(1994) 24, 131-138).

When antibody is produced in in vitro or in vivo production systems, asdescribed above, DNA encoding the heavy chain (H chain) or the lightchain (L chain) of antibody may be separately integrated into anexpression vector and the hosts are transformed simultaneously, or DNAencoding the H chain and the L chain may be integrated into a singleexpression vector and the host is transformed therewith (seeInternational Patent Application WO 94-11523).

Antibodies for use in the present invention may be antibody fragments ormodified versions thereof as long as they are preferably used. Forexample, as fragments of antibody, there may be mentioned Fab, F(ab′)2,Fv or single-chain Fv (scFv) in which Fv's of H chain and L chain wereligated via a suitable linker. Specifically antibodies are treated withan enzyme, for example, papain or pepsin, to produce antibody fragments,or genes encoding these antibody fragments are constructed, and thenintroduced into an expression vector, which is expressed in a suitablehost cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152,2968-2976; Better, M. and Horwitz, A. H., Methods in Enzymology (1989)178, 476-496, Academic Press, Inc.; Plueckthun, A. and Skerra, A.,Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Lamoyi,E., Methods in Enzymology (1986) 121, 652-663; Rousseaux, J. et al.,Methods in Enzymology (1986) 121, 663-669; Bird, R. E. et al., TIBTECH(1991) 9, 132-137).

scFv can be obtained by ligating the V region of H chain and the Vregion of L chain of antibody. In the scFv, the V region of H chain andthe V region of L chain are preferably ligated via a linker, preferablya peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 5879-5883). The V region of H chain and the V region of Lchain in the scFv may be derived from any of the above-mentionedantibodies. As the peptide linker for ligating the V regions, anysingle-chain peptide comprising, for example, 12-19 amino acid residuesmay be used.

DNA encoding scFv can be obtained using DNA encoding the H chain or theH chain V region of the above antibody and DNA encoding the L chain orthe L chain V region of the above antibody as the template by amplifyingthe portion of the DNA encoding the desired amino acid sequence amongthe above sequences by the PCR technique with the primer pair specifyingthe both ends thereof, and by further amplifying the combination of DNAencoding the peptide linker portion and the primer pair which definesthat both ends of said DNA be ligated to the H chain and the L chain,respectively.

Once DNA encoding scFv are constructed, an expression vector containingthem and a host transformed with said expression vector can be obtainedby the conventional methods, and scFv can be obtained using theresultant host by the conventional methods.

These antibody fragments can be produced by obtaining the gene thereofin a similar manner to that mentioned above and by allowing it to beexpressed in a host. “Antibody” as used in the claim of the presentapplication encompasses these antibody fragments.

As modified antibodies, antibodies associated with various moleculessuch as polyethylene glycol (PEG) can be used. “Antibody” as used in theclaim of the present application encompasses these modified antibodies.These modified antibodies can be obtained by chemically modifying theantibodies thus obtained. These methods have already been established inthe art.

Antibodies produced and expressed as described above can be separatedfrom the inside or outside of the host cell and then may be purified tohomogeneity. Separation and purification of the antibody for use in thepresent invention may be accomplished by affinity chromatography. As thecolumn used for such affinity chromatography, there can be mentionedProtein A column and Protein G column. Examples of the carriers used inthe Protein A column are Hyper D, POROS, Sepharose F. F. and the like.Alternatively, methods for separation and purification conventionallyused for proteins can be used without any limitation. Separation andpurification of the antibody for use in the present invention may beaccomplished by combining, as appropriate, chromatography other than theabove-mentioned affinity chromatography, filtration, ultrafiltration,salting-out, dialysis and the like. Chromatography includes, forexample, ion exchange chromatography, hydrophobic chromatography,gel-filtration and the like. These chromatographies can be applied intoHPLC (High Performance Liquid Chromatography). Alternatively,reverse-phase HPLC can be used.

The concentration of antibody obtained above can be determined by themeasurement of absorbance or by the enzyme-linked immunosorbent assay(ELISA) and the like. Thus, when absorbance measurement is employed, theantibody for use in the present invention or a sample containing theantibody is appropriately diluted with PBS(−) and then the absorbance ismeasured at 280 nm, followed by calculation using the absorptioncoefficient of 1.35 OD at 1 mg/ml. When the ELISA method is used,measurement is conducted as follows. Thus, 100 μl of goat anti-human IgG(manufactured by TAGO) diluted to 1 μg/ml in 0.1 M bicarbonate buffer,pH 9.6, is added to a 96-well plate (manufactured by Nunc), and isincubated overnight at 4° C. to immobilize the antibody.

After blocking, 100 μl each of appropriately diluted antibody for use inthe present invention or a sample containing the antibody, or 100 μl ofhuman IgG (manufactured by CAPPEL) as the standard is added, andincubated at room temperature for 1 hour. After washing, 100 μl of5000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody(manufactured by BIO SOURCE) is added, and incubated at room temperaturefor 1 hour. After washing, the substrate solution is added andincubated, followed by the measurement of absorbance at 405 nm using theMICROPLATE READER Model 3550, (manufactured by Bio-Rad) to calculate theconcentration of the desired antibody.

(VI) Pharmaceutical Composition Containing the Antibody of the PresentInvention

The invention also provides an agent for the prophylaxis and/ortreatment of inflammatory bowel diseases (IBD), multiple sclerosis,psoriasis, or hematological cancers. The term “treatment” includes notonly complete cure but also amelioration of a symptom. It is known thatCD81 is overexpressed in an IBD patient and an anti-CD81 antibody isuseful for preventing, ameliorating or treating IBD. Herein,“inflammatory bowel diseases (IBD)” means diseases including ulcerativecolitis and Crohn's disease.

Since the present inventors obtained new findings that the anti-CD81antibodies suppressed T cell migration, the antibody of the presentinvention is also useful for preventing, improving or treating a diseaseassociated with T cell migration such as multiple sclerosis andpsoriasis.

Since the antibody of the present invention does not enhance theproduction of cytokines such as interleukin-2 from T cells, thepharmaceutical composition containing the antibody does not causeadverse side effects due to cytokine overproduction. The effect on thecytokine production in lymphocytes can be determined by a known method,for example, the quantification of cytokines in culture supernatants oflymphocytes cultured under various conditions using ELISA orintracellular cytokine staining of the lymphocytes after treatment, asdescribed in Nature Immunology, VOLUME 8 NUMBER 9 September 2007, 942.

The present inventors clarified a cytotoxic effect of the antibody ofthe present invention on cancer cells derived from patients withhematological cancer, and found that the antibody of the presentinvention is useful as a prophylactic, ameliorating or therapeutic agentfor hematological cancers. According to the WHO Classification,hematological cancers are classified into leukemias, malignant lymphoma,multiple myeloma, and myelodysplastic syndrome. Leukemias are furtherclassified into acute myelogenous leukemia, acute lymphatic leukemia,chronic myelogenous leukemia, and chronic lymphatic leukemia. Malignantlymphomas are classified into Hodgkin's lymphoma and non-Hodgkin'slymphoma.

Acute myelogenous leukemia is the most prevalent type of adult leukemia.Although acute lymphatic leukemia is relatively prevalent in children,it occurs at a certain incidence in adults as well. Chronic myelogenousleukemia is a disease for which therapeutic outcomes have improveddramatically in recent years. Chronic lymphatic leukemia is a type ofleukemia relatively prevalent in Europe and the US, but not lessprevalent in Japanese. Of malignant lymphomas, non-Hodgkin's lymphomasinclude adult T cell lymphoma, lymphoblastic lymphoma, diffuselarge-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, MALTlymphoma, peripheral T cell lymphoma, mantle cell lymphoma and the like.MALT lymphoma and follicular lymphoma are tumors of low malignancy thatadvances so slowly that no major advance occurs during many years. Incontrast, Burkitt's lymphoma, lymphoblastic lymphoma and adult T celllymphoma are tumors of extremely high malignancy that aggravate week byweek. Other types positioned therebetween are peripheral T celllymphoma, diffuse large-cell lymphoma, and mantle cell lymphoma. Thesetumors advance month by month. Multiple myeloma is a type of tumor inthe stage of plasma cells resulting from maturation of B lymphocytes toproduce immunoglobulins. Myelodysplastic syndrome generically refers toa class of diseases characterized by morphological and functionalabnormalities of bone marrow stem cells.

Available treatments for hematological cancers include chemotherapies,radiotherapies, molecule-targeting treatments, and high-dosechemotherapies in combination with hematopoietic stem celltransplantation. While pediatric acute lymphatic leukemia is treatablewith a long survival rate of 80%, adult acute lymphatic leukemia has alow long survival rate of 15-35%, although complete remission isachieved in 60-80% of the patients. The therapeutic outcomes for chroniclymphatic leukemia have been improving since the advent of Gleevec(imatinib mesylate). Improved therapeutic outcomes have been noted inHodgkin's lymphoma and non-Hodgkin's lymphoma of low or moderatemalignancy. Meanwhile, in adult T cell lymphoma, various therapies failto produce improved therapeutic outcomes, the median survival time beingabout 1 year (IGAKU NO AYUMI, Vol. 212, No. 5, pp. 461-466, 2005).Lymphoblastic lymphoma is a set of diseases classified as non-Hodgkin'slymphoma of high malignancy. For Burkitt's lymphoma, the prognosis hasbeen improved by short-time high-dose chemotherapy; the 2-year survivalrate is currently about 70% or more, demonstrating improved therapeuticoutcomes (Magrath, I. et al., J. Clin. Oncol., 14:925-943, 1996; Mead,G. M. et al., Ann. Oncol., 13:1264-1274, 2002); however, the 3-yearsurvival rate remains low at 49%, so that a further improvement in thetherapeutic outcomes is needed (Thomas D. A. et al., J. Clin. Oncol.,17:2461-2470, 1999). In diffuse large-cell lymphoma, a combinationtherapy with an anti-CD20 antibody (rituximab) and CHOP therapy(cyclophosphamide, doxorubicin, vincristine, prednisolone) is becoming astandard treatment with improved therapeutic outcomes for young low-riskpatients in the progression stage. However, for young high-risk patientsin the progression stage, no therapy surpassing CHOP therapy isavailable. Regarding the treatment of multiple myeloma, anticanceragents are somewhat effective, but this disease is highly malignant sothat the treatment is not as effective as for leukemia and lymphoma. Formyelodysplastic syndrome, the 5- to 10-year survival rate is about30-40%. Hematological cancers of high malignancy for which furtherimprovements in the therapeutic efficacy are expected include acutemyelogenous leukemia, acute lymphatic leukemia, lymphoblastic lymphoma,diffuse large-cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma,peripheral T cell lymphoma, adult T cell lymphoma, multiple myeloma,myelodysplastic syndrome and the like.

As stated above, no therapeutic methods or drugs are available withsatisfactory therapeutic efficacy for hematological cancers of moderateto high malignancy; there is a demand for a novel therapeutic method anddrug. It was found that anti-CD81 antibodies, whose potential astherapeutic drugs for hematological cancers has been unclear so far,have cytotoxic effects on some types of hematological cancers for whichthe therapeutic efficacy is lacked, i.e., Jurkat cells (a cancer cellline derived from a patient with acute lymphatic leukemia) and Ramoscells (a cancer cell line derived from a patient with Burkitt'slymphoma), based on their complement-dependent cytotoxicity. The findingthat the anti-CD81 antibody exhibits a cytotoxic effect on cancer cellswas not known so far.

Accordingly, the present invention also provides a prophylactic,ameliorating or therapeutic agent for hematological cancers with theantibody of the present invention as an active ingredient, preferably aprophylactic, ameliorating or therapeutic agent for hematologicalcancers of high malignancy.

The present invention is applicable not only to hematological cancers,but also to any type of cancer cell that expresses CD81. Therefore,drugs with the antibody of the present invention as an active ingredientare effective as a prophylactic, ameliorating or therapeutic agent forcancers caused by CD81-expressing cancer cells.

The agent containing the anti-CD81 antibody as an active ingredient isitself administered as an agent formulated by a known manufacturingpharmaceutical method. For example, it can be used in the form of aninjection solution of a sterile solution or suspension with water orother pharmaceutically acceptable liquids. Further, it is considered tobe formulated by being properly mixed with pharmacologically acceptablecarriers or media, such as sterile water, a physiological salinesolution, an emulsifying agent, a suspending agent, a surfactant, astabilizer, a vehicle and a preservative in the unit dosage formrequired for formulation, which is generally approved. The amount of theactive ingredient in these pharmaceutical preparations is adjusted suchthat an appropriate volume in the prescribed range is obtained.

The sterile composition for injection can be formed according toordinary formulation using a vehicle such as distilled water forinjection. Examples of the aqueous solution for injection include aphysiological saline solution and isotonic solutions containing glucoseand other auxiliaries such as D-sorbitol, D-mannose, D-mannitol andsodium chloride. An appropriate solubilizer, for example, an alcoholsuch as ethanol polyalcohol such as propylene glycol or polyethyleneglycol, a nonionic surfactant such as polysorbate 80™ or HCO-50 may beused in combination.

Examples of oil include sesame oil and soybean oil, and benzyl benzoateor benzyl alcohol may be used in combination as a solubilizer. Abuffering agent such as a phosphate buffer or a sodium acetate buffer, asoothing agent such as procaine hydrochloride, a stabilizer such asbenzyl alcohol, phenol and an antioxidant may be incorporated. Thethus-formed injection solution is usually filled in an appropriateampule.

Regarding the agent containing the anti-CD81 antibody as an activeingredient, both of the oral administration and the parenteraladministration are possible. The parenteral administration ispreferable. Specific examples thereof include an injection solutiondosage form, a transnasal dosage form, a transpulmonary dosage form, apercutaneous dosage form and the like. Regarding examples of theinjection solution dosage form, systemic or local administration can beconducted by intravenous injection, intramuscular injection,intraperitoneal injection, subcutaneous injection or the like.

The dose can properly be selected depending on the age and condition ofpatients. For example, the dose can be selected in the range of from0.0001 mg to 1,000 mg per kilogram of the body weight for oneadministration. Alternatively, the dose can be selected in the range offrom 0.001 to 100,000 mg/body for a patient. However, the therapeuticagent of the invention is not limited by these doses.

The present invention is explained in more detail in the following byreferring to Examples, which are not to be construed as limitative.

EXAMPLES Example 1 Preparation of Human CD81 Antibody (1) Selection ofHuman Anti-Human CD81 Antibody Fragment

A complete human antibody library n-CoDeR (Nature Biotechnol. 18:852-856, 2000; WO98/32845) was screened for specific binding with humanCD81, using human CD81 protein and human CD9 protein having a homologywith CD81, or human CD81-expressing and -non-expressing cells to obtainhuman antibody fragments (scFv) specifically binding with human CD81.First, phages bound to biotinylated CD81 protein in the presence ofcompetitive CD9 protein were recovered using streptoavidin-labeledmagnetic beads. Unbound phages were washed out and the bound phages wereeluted by trypsin treatment. Panning was carried out three times, andthe selected phage library was expressed as an antibody fragment in E.coli. Antibody fragments that bind to CD81-expressing cells but do notbind to CD81-non-expressing cells were screened out using FMAT(Fluorometric Microvolume Assay Technology). FMAT is performed by addingantibody fragments to the cells immobilized on the wells of 384-wellculture plate, and detecting its binding using a fluorescent-labeledanti-scFv antibody. As a result of the screening, two humanCD81-specific human antibody fragments were selected. The nucleotidesequences (scFv-002-A07: SEQ ID NO:33, scFv-005-C01: SEQ ID NO:35) andthe deduced amino acid sequences (scFv-002-A07: SEQ ID NO:34,scFv-005-C01: SEQ ID NO:36) of these fragments were determined bysequencing.

(2) Production of Anti-Human CD81 Antibody

Using the sequence information of the fragments obtained (1) above,anti-human CD81 antibody genes (heavy chain variable region (V_(H)): SEQID NO:9 (002-A07); SEQ ID NO:19 (005-C01) and light chain variableregion (V_(L)): SEQ ID NO:7 (002-A07); SEQ ID NO:17 (005-C01)) wereprepared by a known method (Nat Biotechnol., 18, 852-856, 2000),amplified by PCR using a set of primers having BsmI and NheI recognitionsites. The amplified fragments were digested with BsmI and NheI andsubcloned into pCEP4 (Invitrogen)-derived vectors. In the heavy chainexpression vector, a genomic region of mutant human γ4 chain wherein the228th Ser is substituted with Pro (S228P) is inserted, and in the lightchain expression vector, a genomic region of human λ chain is inserted.Thus constructed heavy and light chain expression vectors wereco-transfected into HEK293-EBNA cells (ATCC-CRL-10852) using FuGene6(Roche) according to the manufacture's protocol and the cells werecultured. After 6 days culture, the IgG4 antibodies were purified fromthe culture supernatants. Briefly, the culture solution was centrifugedand the supernatant was subjected to protein A chromatography. Thepurified material was dialyzed to 10 mM NaP buffer (0.15 M NaCl, pH6.5). The 002-A07 IgG4 antibody thus obtained has the heavy chainconsisting of the amino acid sequence shown in SEQ ID NO:28 and thelight chain consisting of the amino acid sequence shown in SEQ ID NO:26.The 005-C01 IgG4 antibody thus obtained has the heavy chain consistingof the amino acid sequence shown in SEQ ID NO:32 and the light chainconsisting of the amino acid sequence shown in SEQ ID NO:30.

Experimental Example 1 Binding Activity of Anti-CD81 Antibody onCD81-Expressing Cells (1) Cell Preparation

Based on known information (JOURNAL OF VIROLOGY., 79, 4316-4328, 2005),Jurkat E6.1 cells and human PBMC (Peripheral Blood Mononuclear cells)were used as human CD81-expressing cells to confirm binding of theantibodies of the present invention obtained in Example 1 to the humanCD81-expressing cells. Jurkat E6.1 cells were purchased from EuropeanCollection of Cell Cultures (ECACC) (Cat No. 88042803). Human PBMC werepurchased from KAC Co., Ltd. (KAC). These cells were collected bycentrifugation (4,000 rpm, 3 min, 4° C.) and suspended in FACS stainingbuffer (phosphate buffer, 0.09% sodium azide, 1% bovine serum albumin(BSA: Hyclone Laboratories)). The number of viable cells was countedusing trypan blue and 10⁶ cells/100 μl were added in an eppendorf tube.

(2) Cell Staining

To the cells prepared in (1) above was added the anti-human CD81antibody (002-A07 or 005-C01) prepared in Example 1 or a human IgG4(Acris) as a control (0.1 μg), and the cells were incubated for 20 minat 4° C. After washing the cells with FACS staining buffer, PE(phycoerythrin)-labeled anti-human IgG4 antibody (Beckman coulter; 0.05μg) was added to the cells and incubated for 15 min at 4° C. Afterwashing with FACS staining buffer twice, the cells were centrifuged(4,000 rpm, 3 min, 4° C.), the supernatant was removed and theprecipitated cells were fixed with BD Cytefix/Cyteperm buffer (BDBiosciences). After washing, buffer was changed to PBS (NACALAI TESQUE,INC.). The binding ratio (%) of the anti-human CD81 antibody to thecells was analyzed by flow cytometry using FACS Calibur (BD Biosciences)and calculated as the percentage of cells having a PE-derivedfluorescent intensity greater than that observed when the control IgG4is used. The results are shown in Table 1. 002-A07 and 005-C01 stronglybound to Jurkat E6.1 cell and human PBMC.

TABLE 1 Binding of anti-human CD81 antibodies to Jurkat and PBMC. PBMCJurkat Binding ratio to cells (%) control IgG4 1 1 002-A07 99 100005-C01 98 100 The data presented are representative of three individualexperiments.

Experimental Example 2 Suppressive Effect of Anti-Human CD81 Antibody onChemotaxis of Jurakat Cells (1) Subculture of Jurkat Cells

Jurkat E6.1 cells (purchased from ECACC: Cat No. 88042803) weremaintained in RPMI1640 medium (GIBCO) supplemented with 10% fetal calfserum (FCS: Hyclone Laboratories) in humidified incubators in 5% CO₂ at37° C. Cell density was kept between 2×10⁵ and 1×10⁶ cells/ml bydiluting with the medium.

(2) Antibody Treatment

Jurkat E6.1 cells were suspended at 4×10⁶ cells/ml in chemotaxis medium(RPMI1640 (GIBCO) containing 0.5% BSA (SIGMA), 50 mM HEPES (GIBCO)). Theanti-human CD81 antibody (002-A07 or 005-C01) or control human IgG4(Acris Antibodies GmbH) was added to the cells at the finalconcentrations described in Table 2. The cells were incubated for 2hours in humidified incubators in 5% CO₂ at 37° C.

(3) chemotaxis Assay

Chemotaxis was examined using a 96-well chemotaxis chamber (Corning Inc,5 μm pore size). The lower wells were filled with 235 μl of chemotaxismedium in the presence of 10 ng/ml SDF-1 (PEPROTECH). 75 μl ofpreincubated Jurkat cells with or without anti-human CD81 antibody wereloaded onto the upper wells and incubated for 2 hours in humidifiedincubators in 5% CO₂ at 37° C. After incubation, 50 μl of lower wellcell suspension was transferred to 96 well black plate (Corning Inc) andadded 50 μl of ATP lite (Perkin Elmer). The number of the migrated cellswas calculated by measuring luminescence intensity using Envision 2102multilabelreader (Perkin Elmer).

(4) Analysis of Chemotaxis Assay

Percent Chemotaxis was calculated as the percentage of the number of themigrated Jurkat cells when the anti-human CD81 antibody was added, tothe number of the migrated Jurkat cells when control IgG4 was added. Asa result, both anti-human CD81 antibodies inhibited chemotaxis of Jurkatcells (Table 2). Since the suppressing effect of 002-A07 and 005-C01 onT cell migration of Jurkat cells was observed, it was confirmed that theanti-CD81 antibodies (002-A07 and 005-C01) can be a therapeutic agent ofIBD.

TABLE 2 Suppressive effect of 002-A07 and 005- C01 on chemotaxis ofJurkat cells Jurkat cell chemotaxis (%) antibody conc. (μg/ml) 1 0.5 0.20.04 0.005 control IgG4 100 100 100 100 100 002-A07 18 9 20 51 95005-A01 20 42 44 82 102 The data presented are average value of threeindividual experiments.

Experimental Example 3 Suppressive Effect of Anti-Human CD81 Antibody onChemotaxis of Human PBMC (Peripheral Blood Mononuclear Cells)

Suppressive effect of anti-human CD81 antibodies on chemotaxis of humanPBMC was examined according to the method described in Biologicals, 2007October; 35(4):227-33, Epub 2007 Aug. 28.

(1) Cell Preparation

Human PBMC was purchased from KAC. According to the attached informationfrom KAC, 60% of the human PBMC used was T cells (CD3-positive cells).The human PBMC were maintained in RPMI1640 medium (GIBCO) supplementedwith 10% fetal calf serum (FCS: Hyclone Laboratories) in humidifiedincubators in 5% CO₂ at 37° C. The cells (1×10⁶ cells/ml) were incubatedwith 5 μg/ml PHA (phytohemagglutinin, Wako) and 50 ng/ml humanrecombinant IL-2 (R&D system) for 4 days in humidified incubators in 5%CO₂ at 37° C.

(2) Antibody Treatment

Human PBMC were suspended at 4×10⁶ cells/ml in chemotaxis medium(RPMI1640 (GIBCO) containing 0.5% BSA (SIGMA), 50 mM HEPES (GIBCO)). Theanti-human CD81 antibody (002-A07 or 005-C01) or control human IgG4(Acris Antibodies GmbH) was added to the cells at the concentrationsdescribed in Table 3. The cells were incubated for 2 hours in humidifiedincubators in 5% CO₂ at 37° C.

(3) Chemotaxis Assay

Chemotaxis was examined using a 96-well chemotaxis chamber (corningcoster, 5 μm pore size). The lower wells were filled with 235 μl ofchemotaxis medium in the presence of 50 ng/ml SDF-1. 75 μl ofpreincubated human PBMC with or without anti-human CD81 antibody wereloaded onto the upper wells and incubated for 2 hours in humidifiedincubators in 5% CO₂ at 37° C. After incubation, 50 μl of lower wellcell suspension was transferred to 96 well black plate (Corning Inc) andadded 50 μl of ATP lite (Perkin Elmer). The number of migrated cells wascalculated by measuring luminescence intensity using Envision 2102multilabelreader (Perkin Elmer).

(4) Analysis of Chemotaxis Assay

Percent Chemotaxis was calculated as the percentage of the number of themigrated human PBMC when the anti-human CD81 antibody was added, to thenumber of the migrated human PBMC when control IgG4 was added. As aresult, both anti-human CD81 antibodies inhibited chemotaxis of humanPBMC (Table 3). Since the suppressing effect of 002-A07 and 005-C01 on Tcell migration of human PBMC containing primary human T cells wasobserved, it was confirmed that the anti-CD81 antibodies (002-A07 and005-C01) can be a therapeutic agent of IBD.

TABLE 3 Suppressive effect of 002-A07 and 005-C01 on chemotaxis of humanPBMC human PBMC chemotaxis (%) antibody conc. (μg/ml) 1 0.5 0.2 0.04control IgG4 100 100 100 100 002-A07 0 3 17 59 005-C01 27 58 63 87

Experimental Example 4 Effects of Anti-Human CD81 Antibody on CytokineProduction in and Cell Proliferation of Human PBMC

The cytokine production in human PBMC was determined according to LifeSci. 2001 Nov. 21; 70(1):81-96.

(1) Cell Culture and Stimulation

Human PBMC was purchased from KAC. According to the attached informationfrom KAC, 60% of the human PBMC used was T cells (CD3-positive cells).The human PBMC were maintained in RPMI1640 medium (GIBCO) supplementedwith 10% fetal calf serum (FCS: Hyclone Laboratories) in humidifiedincubators in 5% CO₂ at 37° C. To 96-well plate (IWAKI) was added theanti-human CD81 antibody (002-A07 or 005-C01) or control IgG4 (AcrisAntibodies GmbH) at the concentrations described in Table 4, and thehuman PBMC (1×10⁵ cells/ml) were added to each well (100 μl/well). Tothe cells were added 1000 ng/well anti CD3 antibody (Clone: OKT3, BioLegend) and 1000 ng/well anti-CD28 antibody (BD Biosciences) (50 μl/welleach) and incubated for 48 hours in humidified incubators in 5% CO₂ at37° C. After incubation, the IL-2 level of supernatants was measured byELISA assay.

(2) IL-2 ELISA Assay

IL-2 concentration produced from human PBMC to which antibody was addedwas measured by human IL-2 ELISA kit (R&D system). ELISA assay wasconducted according to the manufacture's protocol. Percent IL-2production was calculated as the percentage of the IL-2 level producedfrom human PBMC when the anti-human CD81 antibody was added, to the IL-2level produced from human PBMC when control IgG4 was added. The resultsare shown in Table 4.

(3) Alamar Blue Assay

Cell proliferation was measured by Alamar blue (BIOSOURCE). This assaywas conducted according to the manufacture's protocol. Percent cellproliferation was calculated as the percentage of the cell proliferationof human PBMC when the anti-human CD81 antibody was added, to the cellproliferation of human PBMC when control IgG4 was added. The results areshown in Table 5.

TABLE 4 Effect of 002-A07 and 005-C01 on IL-2 production in human PBMChuman PBMC IL-2 production (%) antibody conc. (μg/ml) 5 1 0.2 0.04control IgG4 100 100 100 100 002-A07 123 108 110 98 005-C01 110 100 101104

TABLE 5 Effect of 002-A07 and 005-C01 on cell proliferation of humanPBMC human PBMC cell proliferation (%) antibody conc. (μg/ml) 5 1 0.20.04 control IgG4 100 100 100 100 002-A07 107 104 105 105 005-C01 114102 103 104

These results demonstrate that both anti-human CD81 antibodies do notstimulate cytokine production in or cell proliferation of human PBMC.Thus, 002-A07 and 005-C01 do not affect cytokine production or cellproliferation that is an index for T cell activation. Accordingly, itwas confirmed that the antibodies have no concern for side effect suchas cytokine storm due to cytokine overproduction or immunosuppressiondue to the suppression of T cell function.

Experimental Example 5 Suppressive Effect of Anti-Human CD81 Antibody onChemotaxis of PBMC from IBD Patients (1) Cell Preparation

IBD patient PBMC (purchased from Tissue solution and the like) aremaintained in RPMI1640 medium (GIBCO) supplemented with 10% fetal calfserum (FCS: Hyclone Laboratories) in humidified incubators in 5% CO₂ at37° C. The cells are incubated with or without phytohemagglutinin (PHA),human recombinant IL-2, human TNFα, vasoactive intestinal peptide (VIP),IL-22 and IL-7 for 4 days in humidified incubators in 5% CO₂ at 37° C.

(2) Antibody Treatment

The IBD patient's PBMC are suspended in chemotaxis medium (RPMI1640(GIBCO) containing 0.5% BSA (SIGMA), 50 mM HEPES (GIBCO)). Theanti-human CD81 antibody (002-A07 or 005-C01) or control human IgG4(Acris Antibodies GmbH) is added to the cells at various concentrations.The cells were incubated for 2 hours in humidified incubators in 5% CO₂at 37° C.

(3) Chemotaxis Assay

Chemotaxis is examined using a 96-well chemotaxis chamber (corningcoster, 5 μm pore size). The lower wells are filled with 235 μl ofchemotaxis medium in the presence of SDF-1. 75 μl of preincubated IBDpatient PBMC with or without anti-human CD81 antibody are loaded ontothe upper wells and incubated for 2 hours in humidified incubators in 5%CO₂ at 37° C. After incubation, 50 μl of lower well cell suspension istransferred to 96 well black plate (Corning Inc) and added 50 μl of ATPlite (Perkin Elmer). The number of the migrated cells is calculated bymeasuring luminescence intensity using Envision 2102 multilabelreader(Perkin Elmer).

(4) Analysis of Chemotaxis Assay

Percent chemotaxis is calculated as the percentage of the number of themigrated IBD patient PBMC when the anti-human CD81 antibody is added, tothe number of the migrated IBD patient PBMC when control IgG4 is added.

Experimental Example 6 Effect of Anti-Human CD81 Antibody on CytokineProduction in IBD Patient PBMC (1) Cell Culture and Stimulation

IBD patient's PBMC (purchased from Tissue solution and the like) aremaintained in RPMI1640 medium (GIBCO) supplemented with 10% fetal calfserum (FCS: Hyclone Laboratories) in humidified incubators in 5% CO₂ at37° C. To 96-well plate (IWAKI) is added the anti-human CD81 antibody(002-A07 or 005-C01) or control IgG4 (Acris Antibodies GmbH) at variousconcentrations, and the IBD patient PBMC are added to each well. To thecells are added 1000 ng/well anti CD3 antibody (Clone: OKT3, Bio Legend)and 1000 ng/well anti-CD28 antibody (BD Biosciences) (50 μl/well each)and incubated for 48 hours in humidified incubators in 5% CO₂ at 37° C.After incubation, the IL-2 level of supernatants is measured by ELISAassay.

(2) IL-2 ELISA Assay

IL-2 concentration produced from IBD patient's PBMC to which antibody isadded is measured by human IL-2 ELISA kit (R&D system). ELISA assay isconducted according to the manufacture's protocol. Percent IL-2production is calculated as the percentage of the IL-2 level producedfrom human PBMC when the anti-human CD81 antibody is added, to the IL-2level produced from human PBMC when control IgG4 is added.

Experimental Example 7 Epitope Mapping of Anti-Human CD81 Antibody UsingHuman and Chicken CD81 Chimeras

To identify the epitopes to which each of the anti-human CD81 antibodies(002-A07 and 005-C01) bind, ELISA assays were performed usinghuman-chicken CD81 chimera constructs listed in Table 6. Each constructwas transiently expressed in CHO cells and its cell membrane fractionwas solubilized with detergents and immobilized onto a plate. Theresults are summarized in Table 7.

(1) Expression of Chimera CD81 Proteins

Using human CD81 gene (SEQ ID NO:21) and chicken CD81 gene (SEQ IDNO:23), 10 types of chimera CD81 genes depicted in Table 6 wereconstructed. Each chimera gene was subcloned into pcDNA-DEST40 vector soas to express as a fusion protein having V5 and 6×His tags at theC-terminal. Each chimera construct was transiently expressed in CHOcells with Trans-IT LT-1 (TAKARA BIO, Code MIR2304) according to themanufacture's protocol, and 48 hours after, the membrane fraction wasprepared by following procedure. To the CD81 chimera-expressing CHOcells was added HBS buffer (20 mM Hepes (Invitrogen), 150 mM NaCl(NACALAI TESQUE, INC.)) containing 1% (w/v) n-Octylglucoside (NACALAITESQUE, INC.), and the cells were incubated for 5 min at 4° C. Then, thecell suspension was centrifuged for 20 min at 10000 g and thesupernatant was collected. The protein content in the supernatant wasdetermined with BCA Protein Assay Kit (Thermo Scientific Pierce, code:23225).

(2) ELISA Assay

As control antibodies, human IgG4 antibody (Acris Antibodies GmbH) andmouse IgG antibody were used. Human-chicken chimera protein was capturedvia His tag introduced into the C-terminal by adding the membranefraction (5 μg/100 μL/well) prepared (1) above to Ni NTA His Sorb plate(QIAGEN) in the presence of 1% (w/v) n-Octylglucoside and incubating for3 hours at 4° C. After washing with TEST (TBS, 0.05% (v/v) Tween 20)three times, an anti-human CD81 antibody or a control antibody (50μL/well) was added and incubated for 1 hour at room temperature (RT).After washing, 2000 fold-diluted HRP-labeled anti-human IgG4 antibody(Mouse anti-human IgG₄ HRP clone: HP6023 Beckman Coulter) or 1000fold-diluted HRP-labeled anti-mouse IgG antibody (Invitrogen) (50μL/well) were added. The plate was incubated for 1 hour at RT and washedwith TBST three times. And then, peroxidase activity was determined byTMB One solution (Promega; Code 53025). The reaction was stopped with 2M H₂SO₄ (50 μL) and absorbance at 450 nm was measured.

(3) Interpretation of Results

Human CD81 have two extracellular domains. 002-A07 and 005-C01 bound tohCD81, c80h, h175c and h190c, but did not bind to cCD81, c138h, c156h,c175h, c190h, h80c, h138c and h156c. These results indicate that theepitopes recognized by 002-A07 and 005-C01 antibodies binding to humanCD81 are present between the 80th amino acid residue and 175th aminoacid residue of human CD81 polypeptide.

TABLE 6 human-chicken CD81 chimeras human CD81 region chicken CD81region hCD81 (SEQ ID NO: 22) 1-236 — cCD81 (SEQ ID NO: 24) — 1-237 h80c1-80  81-237  h138c 1-138 139-237  h156c 1-156 158-237  h175c 1-175177-237  h190c 1-190 192-237  c80h 81-236  1-80  c138h 139-236  1-138c156h 157-236  1-157 c175h 176-236  1-176 c190h 191-236  1-191 Eachnumber shows the position of amino acid residues.

TABLE 7 Epitope mapping using human-chicken CD81 chimeras CD81 antigenconstruct hCD81 cCD81 c80h c138h c156h c175h c190h h80c h138c h156ch175c h190c Antibody 002- ◯ X ◯ X X X X X X X ◯ ◯ A07 005- ◯ X ◯ X X X XX X X ◯ ◯ C01 ◯: Absorbance at 450 nm is 100-10% of that in the caseCD81 is hCD81 X: Absorbance at 450 nm is less than 10% of that in thecase CD81 is hCD81

Experimental Example 8 Epitope Mapping of Anti-Human CD81 Antibody UsingAlanine-Scanning

As shown in Experimental Example 7, the epitope region of human CD81 for002-A07 and 005-C01 antibodies was determined. In this Example, in orderto assess the contribution of individual amino acid residues in theepitope region to binding with the anti-human CD81 antibodies andidentify detailed epitope residues, alanine-scanning mutagenesis(Cunningham & Wells, Science 244: 1081-1085 (1989)) was carried out inthe sequence of the epitope region determined in Experimental Example 7.Human CD81 mutants were prepared by site-directed mutagenesis (this workwas outsourced to TAKARA BIO) and subcloned into pcDNA-DEST40 vector.Subsequently, these mutants were expressed in CHO cells and the bindingof 002-A07 and 005-C01 antibodies with individual mutants was assayed inthe same manner as in Experimental Example 7. Relative binding affinitywas calculated as the percentage of binding affinity of 002-A07 or005-C01 antibody with each mutant to binding affinity of 002-A07 or005-C01 antibody with wild type human CD81, and the mutants wereclassified into 3 groups based on the relative binding affinity. Theexpression levels of the individual mutants were corrected by theexpression levels of V5 tag which was inserted into the C-terminal ofthe mutants. The results are summarized in Table 8.

As shown in Experimental Example 7, the anti-human CD81 antibodies002-A07 and 005-C01 recognize the same epitope region that is locatedbetween the 80th and 175th amino acid residues of human CD81. Theresults of alanine scanning indicate that human CD81 has 13 amino acidresidues (V135, D137, A143, H151, G158, T163, A164, L165, S168, V169,L170, N172 and L174) critical for binding with 002-A07 and 17 amino acidresidues (Y127, A130, L131, V135, V136, D137, N142, A143, L154, 6158,T163, L165, S168, V169, L170, K171 and L174) critical for binding with005-C01 within the epitope region, respectively.

TABLE 8 Epitope mapping of anti-human CD81 antibodies by alaninescanning Amino acid residue Wild type Substitutions No. (codon) (codon)002-A07 005-C01 127 Y (TAT) F (TTC) ◯ Δ 130 A (GCC) T (ACC) ◯ Δ 131 L(CTA) A (GCC) ◯ X 135 V (GTC) A (GCC) X X 136 V (GTC) A (GCC) ◯ Δ 137 D(GAT) A (GCC) X X 142 N (AAC) A (GCC) ◯ Δ 143 A (GCC) T (ACC) Δ X 151 H(CAC) A (GCC) Δ ◯ 154 L (CTT) A (GCC) ◯ X 158 G (GGC) S (AGC) X X 163 T(ACT) A (GCC) Δ Δ 164 A (GCT) T (ACC) X ◯ 165 L (TTG) A (GCC) X X 168 S(TCA) A (GCC) Δ X 169 V (GTG) A (GCC) X X 170 L (CTC) A (GCC) Δ X 171 K(AAG) A (GCC) ◯ Δ 172 N (AAC) A (GCC) Δ ◯ 174 L (TTG) A (GCC) Δ X Eachsymbol shows; ◯: relative binding affinity is equal or slightly weakcompared to wild type human CD81 (relative binding affinity is ranged40-100%). Δ: relative binding affinity is significantly weak compared towild type human CD81 (relative binding affinity is ranged 20-40%). X:relative binding affinity is quite weak compared to wild type human CD81(relative binding affinity is ranged under 20%). (Since the 81st to112nd amino acid residues are transmembrane or intracellular domains,alanine mutants were not prepared.)

Experimental Example 9 Epitope Mapping of the Anti-Human CD81 AntibodyUsing Homolog-Scanning

Since alanine-scanning may cause unwanted conformational change andphysicochemical disruptions (Cunningham and Wells, Science 244:1081-1085 (1989)), we conducted homolog-scanning which is other type ofsystematic scanning to further increase the resolution of energeticprofiling of functional epitopes. The homolog-scanning mutagenesis isdesigned to minimize the possibility of structural disruption upon sidechain substitution by introducing a substitutional group so as tomaintain the structure and function of protein (Protein Science (2005),14:2405-2413).

As shown in Experimental Example 8, human CD81 has 13 and 17 amino acidresidues critical for binding with 002-A07 and 005-C01, respectively,within the epitope region. Homolog mutants were prepared in theseresidues according to the rule described by Protein Science (2005),14:2405-2413). These homolog mutants were expressed in CHO cells and thebinding of the anti-human CD81 antibodies with the individual mutantswas assayed in the same manner as Experimental Example 8.

The results are summarized in Table 9. The homolog-scanning revealedthat human CD81 has 9 amino acid residues critical for binding with002-A07 and 9 amino acid residues critical for binding with 005-C01,respectively, within the epitope region. The 151st, 164th, 168th and172nd residues are specifically critical for binding with 002-A07. Onthe other hand, the 127th, 130th, 143rd and 154th residues arespecifically critical for binding with 005-C01.

TABLE 9 Epitope mapping of anti-human CD81 antibodies byhomolog-scanning Amino acid residue Wild type Substitutions No. (codon)(codon) Homo Ala 002-A07 135 V (GTC) L (CTG) X X 137 D (GAT) E (GAG) X X143 A (GCC) V (GTG) ◯ Δ 151 H (CAC) R (CGC) Δ Δ 158 G (GGC) A (GCC) X X163 T (ACT) S (TCT) ◯ Δ 164 A (GCT) V (GTG) X X 165 L (TTG) I (ATC) ◯ X168 S (TCA) T (ACA) X Δ 169 V (GTG) L (CTG) X X 170 L (CTC) I (ATC) Δ Δ172 N (AAC) Q (CAG) Δ Δ 174 L (TTG) I (ATC) ◯ Δ 005-C01 127 Y (TAT) W(TGG) X Δ 130 A (GCC) V (GTG) X Δ 131 L (CTA) I (ATC) ◯ X 135 V (GTC) L(CTG) X X 136 V (GTC) L (CTG) ◯ Δ 137 D (GAT) E (GAG) X X 142 N (AAC) Q(CAG) ◯ Δ 143 A (GCC) V (GTG) Δ X 154 L (CTT) I (ATC) Δ X 158 G (GGC) A(GCC) X X 163 T (ACT) S (TCT) ◯ Δ 165 L (TTG) I (ATC) ◯ X 168 S (TCA) T(ACA) ◯ X 169 V (GTG) L (CTG) X X 170 L (CTC) I (ATC) Δ X 171 K (AAG) R(AGG) ◯ Δ 174 L (TTG) I (ATC) ◯ X Each symbol shows; ◯: relative bindingaffinity is equal or slightly weak compared to wild type human CD81(relative binding affinity is ranged 40-100%). Δ: relative bindingaffinity is significantly weak compared to wild type human CD81(relative binding affinity is ranged 20-40%). X: relative bindingaffinity is quite weak compared to wild type human CD81 (relativebinding affinity is ranged under 20%). Homo: homolog mutants; Ala:alanine mutants

Example 2 Generating 002-A07 Mutant Antibodies Construction ofGlycosylation Site Mutants

The VL region from n-CoDeR (Registered Trade Mark) clone 002-A07 wasmodified to eliminate the potential N-glycosylation site (amino acidsequence: NLS) located in CDR3. Six VL variants were designed andpurchased from Geneart AG (Regensburg Germany): N113S, N113G, N113Q,S115A, S115G and S115N. These were inserted into an expression vectorcontaining the λ constant region as described in Example 1. The sixvariant light chains were transfected, together with the 002-A07 γ4S228P heavy chain, into HEK293-EBNA cells. Antibodies were expressed andpurified as described in Example 1.

Isolation of Clones from Affinity Maturation

Library Construction

A mutagenized library was created for n-CoDeR (Registered Trade Mark)clone 002-A07. Plasmid DNA was used as template in an error-prone PCRprotocol (Saviranta at al. 1998) where the mutations are introducedrandomly over the entire antibody variable regions. The resultingfragments were ligated into a phagemid vector and electroporated into E.coli HB101F′ (constructed by conjugation of the F′ plasmid into strainHB101 (Invitrogen)) for the construction of a Fab library. The librarywas stored as bacterial glycerol stocks at −80° C. (Saviranta P, PajunenM, Jauria P, Karp M, Pettersson K, MäntsäläP and Lövgren T (1998).“Engineering the steroid-specificity of an anti-17β-estradiol Fab byrandom mutagenesis and competitive phage panning”. Prot Eng 11(2)143-152.)

Phage Display Panning and Screening of Individual Soluble Fab

Phages with Fab display were expressed from the E. coli library usinghelper phage R408 (Stratagene) and purified from the culture supernatantusing PEG precipitation.

CD81-specific clones with improved affinity were isolated by phagedisplay technology. Two parallel panning strategies (A and B), eachconsisting of two consecutive pannings, were used to isolate clones withimproved affinity. In strategy A purified recombinant human CD81 proteincoated on polystyrene beads was used as target. In strategy B Jurkatcells with endogenous expression of human CD81 were used as target.Unbound phages were removed by washing. Target binding Fab-phages wereeluted using trypsin and amplified in E. coli HB101F′.

Phagemid DNA was isolated from the amplified pool of clones afterpanning 2. Gene III was excised by restriction enzyme digestion followedby re-ligation resulting in a Fab expressing plasmid pool. Plasmids weretransformed into E. coli TOP10 (Invitrogen) and transformants expressingindividual soluble Fab selected on antibiotic-containing agar plates.Individual bacterial colonies were transferred from agar plates tomicrotiter plates for expression of soluble Fab with C-terminal His-tag.

Primary screening was performed using an ELISA set-up with sequentialaddition of the following reagents: 1) coating of monoclonal anti-Hisantibody; 2) His-tagged Fab from affinity maturation; 3) FLAG-taggedrecombinant human CD81 protein; 4) AP-conjugated anti-FLAG antibody; 5)Luminescence substrate. Clones with the highest activity in the primaryscreening were cherry picked to new microtiter plates and re-expressed.A secondary screening was performed with the same ELISA set-up asdescribed for the primary screening. Clones with improved bindingcompared to the parent clone 002-A07 in Fab format were analyzed by DNAsequencing. Unique Fab clones were purified from E. coli periplasm usingNi-NTA chromatography. Affinity ranking of purified Fab clones wasperformed using flow cytometry with Jurkat cells and Biacore withimmobilized recombinant human CD81 protein.

Production of IgG4-S228P

Clones with improved affinity compared to the parent clone wereconverted to IgG4-5228P format, expressed and purified as described inExample 1.

The sequences of the 002-A07 mutant antibodies obtained, determined byDNA sequencing, are shown in the sequence listing according to thecorrespondence in the table 10 below.

TABLE 10 SEQ ID NOs representing peptide or DNA sequences of antibodiesgroup 1 2 3 4 5 6 7 8 9 10 11 12 group 25 26 27 28 29 30 31 32 33 34 3536 002- 002- 002- 002- 002- 002- 002- 005- A07 A07 A07 A07 A07 A07 001-002- 002- 002- 002- Antibody A07 C01 N113G N113Q N113S S115A S115G S115NB06 B05 B07 C02 C09 L1CDR 1 11 1 1 1 1 1 1 1 60 1 1 60 peptide L2CDR 212 2 2 2 2 2 2 2 2 2 2 2 peptide L3CDR 3 13 37 40 43 46 49 52 43 3 66 366 peptide H1CDR 4 14 4 4 4 4 4 4 4 4 4 4 4 peptide H2CDR 5 15 5 5 5 5 55 5 5 5 69 5 peptide H3CDR 6 16 6 6 6 6 6 6 55 61 6 70 6 peptide group49 50 51 52 53 54 55 56 57 58 59 60 L-chain 7 17 125 126 127 128 129 130131 133 135 137 139 variable region L-chain 8 18 38 41 44 47 50 53 56 6267 71 75 variable region peptide H-chain 9 19 9 9 9 9 9 9 132 134 136138 136 variable region H-chain 10 20 10 10 10 10 10 10 57 63 10 72 10variable region peptide group 73 74 75 76 77 78 79 80 81 82 83 84L-chain 25 29 DNA L-chain 26 30 39 42 45 48 51 54 58 64 68 73 76 peptideH-chain 27 31 DNA H-chain 28 32 28 28 28 28 28 28 59 65 28 74 28 peptidescFv DNA 33 35 scFv 34 36 peptide group 13 14 15 16 17 18 19 20 21 22 2324 group 37 38 39 40 41 42 43 44 45 46 47 48 002- 002- 002- 002- 002-002- 002- 002- 003- 003- 003- 003- Antibody D03 D08 D10 F01 F05 F07 H02H03 A10 A11 D07 F08 L1CDR 1 80 1 1 1 98 60 1 1 1 1 1 peptide L2CDR 2 2 22 2 2 2 2 2 2 2 2 peptide L3CDR 3 3 66 90 52 3 3 90 3 66 3 90 peptideH1CDR 77 4 4 4 4 4 4 4 4 110 4 4 peptide H2CDR 5 81 5 5 5 5 5 5 5 5 5120 peptide H3CDR 6 6 6 6 93 99 99 6 55 6 115 6 peptide group 61 62 6364 65 66 67 68 69 70 71 72 L-chain 7 141 143 145 146 148 150 151 153 155157 159 variable region L-chain 8 82 86 91 94 100 104 106 108 111 116121 variable region peptide H-chain 140 142 144 136 147 149 149 152 154156 158 160 variable region H-chain 78 83 87 10 95 101 101 10 57 112 117122 variable region peptide group 85 86 87 88 89 90 91 92 93 94 95 96L-chain DNA L-chain 26 84 88 92 96 102 105 107 109 113 118 123 peptideH-chain DNA H-chain 79 85 89 28 97 103 103 28 59 114 119 124 peptidescFv DNA scFv peptide

Experimental Example 10 Suppressive Effects of 002-A07 Mutant Antibodieson Chemotaxis of Jurkat Cells

This experiment was performed in the same manner as Experimental Example2. As a result, all 002-A07 mutant antibodies exhibited suppressiveeffects on chemotaxis of Jurkat cells (a human T cell line) (Table 11).

TABLE 11 Suppressive effects of 002-A07 mutant antibodies on chemotaxisof Jurkat cells 10 1 0.1 0.01 0.001 antibody μg/mL μg/mL μg/mL μg/mLμg/mL control IgG4 100 100 100 100 100 002-A07 3 −2 21 93 90 001-B06 3 335 91 102 002-B05 −10 −1 10 78 84 002-B07 −9 4 8 87 92 002-C02 −4 −5 31104 106 002-C09 11 −1 15 82 101 002-D03 16 14 25 91 105 002-D08 14 19 67105 101 002-D10 10 6 34 87 97 002-F01 9 4 35 87 89 002-F05 7 5 27 79 102002-F07 15 13 38 88 101 002-H02 −2 −10 −1 85 87 002-H03 −3 0 13 89 106003-A10 3 3 15 93 104 003-A11 2 1 16 86 91 003-D07 −9 −5 42 79 87003-F08 −4 −3 21 79 80 002-A07 N113G 7 13 38 101 99 002-A07 N113Q 12 1337 99 104 002-A07 N113S 12 13 40 101 99 002-A07 S115A −9 −4 32 78 82002-A07 S115G −3 −9 13 74 84 002-A07 S115N −9 −11 −4 69 79

Experimental Example 11 Effects of 002-A07 Mutant Antibodies on CytokineProduction by And Cell Proliferation of Human PBMC

This experiment was performed in the same manner as Experimental Example4. 5A6, a commercially available mouse anti-human CD81 antibody (SantaCruz Co.) that enhances IL-2 production while suppressing T cellmigration, was used for control. As a result, none of the 002-A07 mutantantibodies enhanced IL-2 production by human PBMCs (Table 12), nor didthey have any noticeable effect on the cell proliferation (Table 13).

TABLE 12 Effects of 002-A07 mutant antibodies on IL-2 production byhuman PBMC 10 1 antibody μg/mL μg/mL mouse IgG 100 100 5A6 320 179 humanIgG4 100 100 002-A07 109 111 001-B06 174 130 002-B05 59 109 002-B07 67100 002-C02 57 90 002-C09 77 95 002-D03 49 127 002-D08 101 140 002-D1096 169 002-F01 101 188 002-F05 92 187 002-F07 78 224 002-H02 61 156002-H03 82 124 003-A10 109 114 003-A11 84 128 003-D07 78 89 003-F08 98108 002-A07 N113G 117 114 002-A07 N113Q 101 92 002-A07 N113S 80 92002-A07 S115A 76 96 002-A07 S115G 115 91 002-A07 S115N 120 118

TABLE 13 Effects of 002-A07 mutant antibodies on cell proliferation ofhuman PBMC 10 1 antibody μg/mL μg/mL mouse IgG 100 100 5A6 91 95 humanIgG4 100 100 002-A07 102 101 001-B06 125 102 002-B05 121 103 002-B07 111106 002-C02 113 109 002-C09 136 105 002-D03 114 107 002-D08 107 97002-D10 112 97 002-F01 109 100 002-F05 106 103 002-F07 108 97 002-H02105 96 002-H03 118 103 003-A10 112 110 003-A11 118 110 003-D07 121 97003-F08 120 110 002-A07 N113G 117 103 002-A07 N113Q 123 103 002-A07N113S 119 108 002-A07 S115A 126 108 002-A07 S115G 145 97 002-A07 S115N121 87

Experimental Example 12 Epitope Mapping of 002-A07 Mutant AntibodiesUsing Alanine Scanning

This experiment was performed in the same manner as Experimental Example8. Since the 1st to 43rd, 63rd to 112th, 202nd and subsequent residuesare transmembrane or intracellular domains [Levy, S. et al., Annu. Rev.Immunol. (1998) 16, 89-109], no alanine mutant was generated. Since the156th, 157th, 175th, and 190th residues are cysteine residues, noalanine mutant was generated. The relative binding affinity for alaninemutants not listed in Table 14 was equal or slightly weak in all 002-A07mutant antibodies compared to wild type human CD81 (relative bindingaffinity is ranged 40-100).

TABLE 14 Epitope mapping of 002-A07 mutant antibodies by alaninescanning substitution Y127F V135A D137A A143T H151A G158S T163A A164TL165A 002-A07 ◯ X X Δ X X X X X 002-A07 ◯ X X ◯ X X X X X N113G 002-A07◯ X X ◯ X X X X X N113Q 002-A07 ◯ X X ◯ X X X X X N113S 002-A07 Δ X X ΔX X X X X S115A 002-A07 ◯ X X ◯ Δ X X X X S115G 002-A07 ◯ X Δ ◯ ◯ Δ ◯ XX S115N 001-B06 ◯ Δ ◯ ◯ ◯ ◯ ◯ Δ Δ 002-B05 ◯ X X ◯ Δ X Δ X X 002-B07 ◯ XΔ ◯ ◯ Δ ◯ X X 002-C02 ◯ X X ◯ ◯ Δ ◯ X X 002-C09 ◯ X Δ ◯ ◯ Δ ◯ X X002-D03 ◯ X X ◯ ◯ Δ ◯ X X 002-D08 ◯ X Δ ◯ ◯ Δ ◯ X X 002-D10 ◯ X ◯ ◯ ◯ ◯◯ X X 002-F01 ◯ X Δ ◯ ◯ Δ ◯ X X 002-F05 ◯ X Δ ◯ ◯ Δ ◯ X X 002-F07 ◯ X Δ◯ ◯ Δ ◯ X X 002-H02 ◯ X Δ ◯ ◯ ◯ ◯ X X 002-H03 ◯ X ◯ ◯ ◯ ◯ ◯ X X 003-A10◯ Δ ◯ ◯ ◯ ◯ ◯ Δ Δ 003-A11 ◯ X Δ ◯ ◯ Δ ◯ X X 003-D07 ◯ X X ◯ Δ X Δ X X003-F08 ◯ X X ◯ Δ X Δ X X substitution S168A V169A L170A K171A N172AL174A I194A 002-A07 Δ X Δ Δ Δ Δ X 002-A07 Δ X X ◯ ◯ ◯ X N113G 002-A07 ΔX Δ ◯ ◯ ◯ X N113Q 002-A07 Δ X X ◯ ◯ ◯ X N113S 002-A07 Δ X X ◯ ◯ ◯ XS115A 002-A07 Δ X Δ ◯ Δ ◯ X S115G 002-A07 Δ X ◯ ◯ ◯ ◯ Δ S115N 001-B06 ◯X ◯ ◯ ◯ ◯ Δ 002-B05 ◯ X Δ ◯ ◯ ◯ Δ 002-B07 ◯ X ◯ ◯ ◯ ◯ Δ 002-C02 ◯ X ◯ ◯◯ ◯ Δ 002-C09 ◯ X ◯ ◯ ◯ ◯ Δ 002-D03 ◯ X ◯ ◯ ◯ ◯ Δ 002-D08 ◯ X Δ ◯ ◯ ◯ ◯002-D10 ◯ X ◯ ◯ ◯ ◯ Δ 002-F01 Δ X Δ ◯ ◯ ◯ X 002-F05 X X ◯ ◯ ◯ ◯ X002-F07 ◯ X ◯ ◯ Δ Δ X 002-H02 ◯ X ◯ ◯ ◯ ◯ Δ 002-H03 ◯ X ◯ ◯ ◯ ◯ Δ003-A10 ◯ X ◯ Δ X Δ Δ 003-A11 ◯ X ◯ ◯ Δ Δ X 003-D07 ◯ X Δ ◯ X Δ X003-F08 ◯ X Δ ◯ Δ Δ Δ Each symbol shows; ◯: relative binding affinity isequal or slightly weak compared to wild type human CD81 (relativebinding affinity is ranged 40-100%). Δ: relative binding affinity issignificantly weak compared to wild type human CD81 (relative bindingaffinity is ranged 20-40%). X: relative binding affinity is quite weakcompared to wild type human CD81 (relative binding affinity is rangedunder 20%).

Experimental Example 13 Epitope Mapping of 002-A07 Mutant AntibodiesUsing Homolog-Scanning

Amino acid residues thought to be important to the binding of 002-A07mutant antibodies, judging from the results of alanine scanning, weresubjected to homolog-scanning in the same manner as Experimental Example9. As a result, the amino acid residues important to the binding shownbelow were identified respectively for the 002-A07 mutant antibodies. Itwas found that all 002-A07 mutant antibodies bind to the same epitope aswith 002-A07.

-   002-A07 N113G: V135, D137, H151, G158, A164, S168, V169, L170-   002-A07 N113Q: V135, D137, H151, G158, A164, S168, V169, L170-   002-A07 N113S: V135, D137, H151, G158, A164, S168, V169, L170-   002-A07 S115A Y127, V135, D137, A143, H151, G158, A164, S168, V169,    L170-   002-A07 S115G: V135, D137, H151, G158, A164, S168, V169, L170, N172-   002-A07 S115N: V135, D137, A164, S168, V169-   001-B06: A164, V169-   002-B05: V135, D137, H151, 6158, A164, V169, L170-   002-B07: V135, A164, V169-   002-002: V135, D137, A164, V169-   002-009: A164, V169-   002-003: V135, D137, A164, V169-   002-D08: A164, V169-   002-D10: A164, V169-   002-F01: V135, D137, G158, A164, S168, V169-   002-F05: V135, D137, A164, S168, V169-   002-F07: V135, D137, A164, V169-   002-H02: A164, V169-   002-H03: A164, V169-   003-A10: A164, V169-   003-A11: A164, V169-   003-D07: V135, D137, H151, G158, A164, V169-   003-F08: V135, D137, H151, G158, A164, V169, L170

TABLE 15 Epitope mapping of 002-A07 mutant antibodies byhomolog-scanning substitution Y127W V135L D137E A143V H151R G158A T163SA164V L165I 002-A07 — X X ◯ X X ◯ X ◯ 002-A07 — X X — X X ◯ X ◯ N113G002-A07 — X X — X X ◯ X ◯ N113Q 002-A07 — X X — X X ◯ X ◯ N113S 002-A07X X X Δ X X ◯ X ◯ S115A 002-A07 — X X — X X ◯ X ◯ S115G 002-A07 — Δ Δ —— ◯ — X ◯ S115N 001-B06 — ◯ — — — — — Δ ◯ 002-B05 — X Δ — X Δ ◯ X ◯002-B07 — Δ ◯ — — ◯ — X ◯ 002-C02 — Δ X — — ◯ — X ◯ 002-C09 — ◯ ◯ — — ◯— X ◯ 002-D03 — Δ Δ — — ◯ — X ◯ 002-D08 — ◯ ◯ — — ◯ — X ◯ 002-D10 — ◯ —— — — — X ◯ 002-F01 — Δ Δ — — Δ — X ◯ 002-F05 — Δ Δ — — ◯ — X ◯ 002-F07— Δ Δ — — ◯ — X ◯ 002-H02 — ◯ ◯ — — — — X ◯ 002-H03 — ◯ — — — — — X ◯003-A10 — ◯ — — — — — Δ ◯ 003-A11 — ◯ ◯ — — ◯ — X ◯ 003-D07 — X Δ — X Δ◯ X ◯ 003-F08 — X X — X Δ ◯ X ◯ substitution S168T V169L L170I K171RN172Q L174I I194L 002-A07 X X Δ ◯ Δ ◯ ◯ 002-A07 X X Δ — — — ◯ N113G002-A07 X X Δ — — — ◯ N113Q 002-A07 X X Δ — — — ◯ H113S 002-A07 X X Δ —— — ◯ S115A 002-A07 X X Δ — Δ — ◯ S115G 002-A07 X X — — — — ◯ S115N001-B06 — Δ — — — — ◯ 002-B05 — X Δ — — — ◯ 002-B07 — X — — — — ◯002-C02 — X — — — — ◯ 002-C09 — X — — — — ◯ 002-D03 — X — — — — ◯002-D08 — Δ ◯ — — — — 002-D10 — X — — — — ◯ 002-F01 X X ◯ — — — ◯002-F05 X X — — — — ◯ 002-F07 — X — — ◯ ◯ ◯ 002-H02 — X — — — — ◯002-H03 — Δ — — — — ◯ 003-A10 — Δ — ◯ ◯ ◯ ◯ 003-A11 — X — — ◯ ◯ ◯003-D07 — X ◯ — ◯ ◯ ◯ 003-F08 — X Δ — ◯ ◯ ◯ Each symbol shows; ◯:relative binding affinity is equal or slightly weak compared to wildtype human CD81 (relative binding affinity is ranged 40-100%). Δ:relative binding affinity is significantly weak compared to wild typehuman CD81 (relative binding affinity is ranged 20-40%). X: relativebinding affinity is quite weak compared to wild type human CD81(relative binding affinity is ranged under 20%). —: Not tested (sincethe mutant was judged to be unimportant to the binding based on theresults of alanine scanning).

Example 3 Generating Human IgG1-Type Anti-CD81 Antibody

An EcoRI site and an XhoI site were added to each ends of the DNAfragment encodes L-chain peptide sequence of 002-A07 having a human IL-2signal sequence added to the N-terminal portion thereof, and thefragment was inserted into the EcoRI-XhoI site of pcDNA3.1(+)(Invitrogen Co.) (a). Likewise, an EcoRI site and an XhoI site wereadded to each ends of the DNA fragment encodes H-chain peptide sequenceof 002-A07 having a human IL-2 signal sequence added to the N-terminalportion thereof, and the fragment was inserted into the EcoRI-XhoI Siteof pcDNA3.1(+). Furthermore, the DNA fragment was amplified by PCR withthe above-described plasmid incorporating the H-chain gene as thetemplate, using the DNA primers shown below.

5′ side DNA primer (a sequence containing EcoRI site portion):5′-GGTGGAATTCCCACCATGTACAGGATGCAAC-3′ (SEQ ID NO:161)3′ side DNA primer (a sequence containing XhoI site on pFUSE-CHIg-hG1(Invitrogen Co.) and a partial sequence encoding the C-terminal regionof the variable region of the H-chain of 002-A07):5′-TGCACTCGAGACGGTGACCAGTGTACCTTGGCCCC-3′ (SEQ ID NO:162)

After digestion with EcoRI and XhoI, the amplified DNA was inserted intothe EcoRI-XhoI site of pFUSE-CHIg-hG1 to yield a converted-to-IgG1H-chain expression plasmid (b). To prepare a human IgG1-type anti-CD81antibody protein, the plasmids (a) and (b) were transiently introducedinto CHO—S cells, and the cells were subjected to suspension culture.The culture supernatant was recovered, and the human IgG1-type anti-CD81antibody was purified using a Protein A column. The nucleotide and aminoacid sequences of L- and H-chains of the antibody are shown in theSequence Listing.

L-Chain:

-   -   Nucleotide sequence: SEQ ID NO:163    -   Amino acid sequence: SEQ ID NO:164

H-Chain:

-   -   Nucleotide sequence: SEQ ID NO:165    -   Amino acid sequence: SEQ ID NO:166

Experimental Example 14 Binding Activity of Human IgG1-Type Anti-CD81Antibody on Cancer Cells

Binding of a human IgG1-type anti-CD81 antibody to Jurkat E6.1 cells(Cat No. 88042803) derived from a human acute lymphatic leukemia patientand Ramos (RAI) cells (Cat No. EC85030802) derived from a humanBurkitt's lymphoma patient was examined in the same manner asExperimental Example 1, except that human IgG (AbD Serotec Co.) was usedas a control, and that a PE (phycoerythrin)-labeled anti-human Igantibody (Beckman Coulter Co.) was used for the staining. The results ofan analysis using FACS Calibur (BD Biosciences Co.) are shown in Table16. The numerical values in the table are geometric means for FL2 in theFACS Calibur. As a result, human IgG1-type anti-CD81 antibody wasconfirmed as binding to Jurkat cells and Ramos cells.

TABLE 16 Binding of human IgG1-type anti-CD81 antibody to Jurkat cellsand Ramos cells cell Jurkat Ramos Antibody (μg/mL) 10 1 0.1 0.01 10 10.1 0.01 control IgG 8 4 4 4 8 5 4 4 human IgG1- 2310 930 144 32 21291183 712 584 type anti- human CD81 antibody

Experimental Example 15 Cytotoxic Effect (CDC: Complement-DependentCytotoxicity) of Human IgG1-Type Anti-CD81 Antibody on Cancer Cells

After centrifugation at 4,000 rpm (4° C., 3 minutes), Jurkat cells andRamos cells were recovered and suspended in CDC assay buffer (anRPMI1640 medium containing 20 mM Hepes and 0.1% bovine serum albumin).Viable cells were counted using Trypan Blue (GIBCO Co.); the cells weresuspended in the CDC assay buffer to obtain a cell density of 10⁶cells/mL. The cells suspended were dispended to a 96-well cell cultureplate at 50 μL per well; the antibody shown in Table 17 was added at 50μL per well, and the plate was incubated at 37° C. for 30 minutes. A dryrabbit complement (CEDARRLANE Co.) was rehydrated with sterile distilledwater and diluted 10 fold with the CDC assay buffer, after which 50 μLof the dilution was added to each well, and the plate was incubated at37° C. for 2 more hours. A 100 μL aliquot of the culture supernatant wasmixed with 100 μL of the reaction liquid in an LDH assay kit (Cat. No.744934001, Roche Co.), and they were reacted at room temperature for 30minutes, after which absorbance at 490 nm wavelength was measured usinga plate reader. CDC activity was calculated as the percent ratio to theLDH activity value obtained when the cells were completely killed byTriton X-100 treatment. As a result, the complement-dependent cytotoxicactivity of the human IgG1-type anti-CD81 antibody on Jurkat cells andRamos cells was confirmed.

TABLE 17 Cytotoxic effects of human IgG1-type anti-CD81 antibody onJurkat cells cell Jurkat antibody (ng/mL) 10000 1000 100 10 1 0.1control IgG 5.8 4.6 4.1 4.6 3.7 4.7 human IgG1- 84.9 73.3 42.6 6.4 5.96.3 type anti- human CD81 antibody

TABLE 18 Cytotoxic effects of human IgG1-type anti-CD81 antibody onRamos cells cell Ramos antibody (ng/mL) 10000 1000 100 10 1 0.1 controlIgG 0.2 1.7 1.0 1.9 0.4 −1.1 human IgG1- 71.7 71.4 69.4 49.3 26.0 9.9type anti- human CD81 antibody

INDUSTRIAL APPLICABILITY

The anti-human CD81 antibody of the present invention is useful forpreventing, improving or treating inflammatory bowel diseases (IBD),diseases associated with T cell migration such as multiple sclerosis andpsoriasis, or hematological cancer.

1. An isolated anti-human CD81 antibody capable of binding to a peptideregion consisting of the amino acid sequence of the amino acid numbers80 to 175 in the amino acid sequence shown in SEQ ID NO:22.
 2. Theantibody of claim 1, wherein the peptide region consists of the aminoacid sequence of the amino acid numbers 113 to
 175. 3. The antibody ofclaim 1, wherein the binding affinity of the antibody to at least onehuman CD81 variant selected from the group consisting of the following(1) to (13) is less than 40% of that to the human CD81 having the aminoacid sequence shown in SEQ ID NO:22. (1) CD81 variant having the aminoacid sequence shown in SEQ ID NO:22 wherein tyrosine at the amino acidnumber 127 is substituted with phenylalanine or tryptophan; (2) CD81variant having the amino acid sequence shown in SEQ ID NO:22 whereinalanine at the amino acid number 130 is substituted with threonine orvaline; (3) CD81 variant having the amino acid sequence shown in SEQ IDNO:22 wherein valine at the amino acid number 135 is substituted withalanine or leucine; (4) CD81 variant having the amino acid sequenceshown in SEQ ID NO:22 wherein aspartic acid at the amino acid number 137is substituted with alanine or glutamic acid; (5) CD81 variant havingthe amino acid sequence shown in SEQ ID NO:22 wherein alanine at theamino acid number 143 is substituted with threonine or valine; (6) CD81variant having the amino acid sequence shown in SEQ ID NO:22 whereinhistidine at the amino acid number 151 is substituted with alanine orarginine; (7) CD81 variant having the amino acid sequence shown in SEQID NO:22 wherein leucine at the amino acid number 154 is substitutedwith alanine or isoleucine; (8) CD81 variant having the amino acidsequence shown in SEQ ID NO:22 wherein glycine at the amino acid number158 is substituted with alanine or serine; (9) CD81 variant having theamino acid sequence shown in SEQ ID NO:22 wherein alanine at the aminoacid number 164 is substituted with threonine or valine; (10) CD81variant having the amino acid sequence shown in SEQ ID NO:22 whereinserine at the amino acid number 168 is substituted with alanine orthreonine; (11) CD81 variant having the amino acid sequence shown in SEQID NO:22 wherein valine at the amino acid number 169 is substituted withalanine or leucine; (12) CD81 variant having the amino acid sequenceshown in SEQ ID NO:22 wherein leucine at the amino acid number 170 issubstituted with alanine or isoleucine; and (13) CD81 variant having theamino acid sequence shown in SEQ ID NO:22 wherein asparagine at theamino acid number 172 is substituted with alanine or glutamine.
 4. Theantibody of claim 3, wherein the binding affinity of the antibody toeach of the above-identified human CD81 variants (9) and (11) is lessthan 40% of that to the human CD81 having the amino acid sequence shownin SEQ ID NO:22.
 5. An isolated antibody having a binding propertyequivalent to that of the antibody of claim 1, or binding to the humanCD81 having the amino acid sequence shown in SEQ ID NO:22 competitivelywith the antibody of claim
 1. 6. An isolated antibody binding to thehuman CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with the antibody of claim 1, and having a suppressiveeffect of T cell migration.
 7. An isolated anti-human CD81 antibody,which comprises all 6 CDRs described in any one of the following groups1 to
 24. Group 1 (a-1) a CDR comprising the amino acid sequence shown inSEQ ID NO:1, (b-1) a CDR comprising the amino acid sequence shown in SEQID NO:2, (c-1) a CDR comprising the amino acid sequence shown in SEQ IDNO:3, (d-1) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-1) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-1) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 2 (a-2) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-2) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-2) a CDR comprising the amino acid sequence shown in SEQ IDNO:37, (d-2) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-2) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-2) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 3 (a-3) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-3) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-3) a CDR comprising the amino acid sequence shown in SEQ IDNO:40, (d-3) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-3) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-3) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 4 (a-4) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-4) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-4) a CDR comprising the amino acid sequence shown in SEQ IDNO:43, (d-4) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-4) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-4) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 5 (a-5) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-5) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-5) a CDR comprising the amino acid sequence shown in SEQ IDNO:46, (d-5) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-5) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (t-5) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 6 (a-6) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-6) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-6) a CDR comprising the amino acid sequence shown in SEQ IDNO:49, (d-6) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-6) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-6) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 7 (a-7) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-7) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-7) a CDR comprising the amino acid sequence shown in SEQ IDNO:52, (d-7) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-7) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-7) a CDR comprising the amino acid sequence shown in SEQ IDNO:6 Group 8 (a-8) a CDR comprising the amino acid sequence shown in SEQID NO:1, (b-8) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-8) a CDR comprising the amino acid sequence shown in SEQ IDNO:43, (d-8) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-8) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-8) a CDR comprising the amino acid sequence shown in SEQ IDNO:55 Group 9 (a-9) a CDR comprising the amino acid sequence shown inSEQ ID NO:60, (b-9) a CDR comprising the amino acid sequence shown inSEQ ID NO:2, (c-9) a CDR comprising the amino acid sequence shown in SEQID NO:3, (d-9) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-9) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-9) a CDR comprising the amino acid sequence shown in SEQ IDNO:61 Group 10 (a-10) a CDR comprising the amino acid sequence shown inSEQ ID NO:1, (b-10) a CDR comprising the amino acid sequence shown inSEQ ID NO:2, (c-10) a CDR comprising the amino acid sequence shown inSEQ ID NO:66, (d-10) a CDR comprising the amino acid sequence shown inSEQ ID NO:4, (e-10) a CDR comprising the amino acid sequence shown inSEQ ID NO:5, and (f-10) a CDR comprising the amino acid sequence shownin SEQ ID NO:6 Group 11 (a-11) a CDR comprising the amino acid sequenceshown in SEQ ID NO:1, (b-11) a CDR comprising the amino acid sequenceshown in SEQ ID NO:2, (c-11) a CDR comprising the amino acid sequenceshown in SEQ ID NO:3, (d-11) a CDR comprising the amino acid sequenceshown in SEQ ID NO:4, (e-11) a CDR comprising the amino acid sequenceshown in SEQ ID NO:69, and (f-11) a CDR comprising the amino acidsequence shown in SEQ ID NO:70 Group 12 (a-12) a CDR comprising theamino acid sequence shown in SEQ ID NO:60, (b-12) a CDR comprising theamino acid sequence shown in SEQ ID NO:2, (c-12) a CDR comprising theamino acid sequence shown in SEQ ID NO:66, (d-12) a CDR comprising theamino acid sequence shown in SEQ ID NO:4, (e-12) a CDR comprising theamino acid sequence shown in SEQ ID NO:5, and (f-12) a CDR comprisingthe amino acid sequence shown in SEQ ID NO:6 Group 13 (a-13) a CDRcomprising the amino acid sequence shown in SEQ ID NO:1, (b-13) a CDRcomprising the amino acid sequence shown in SEQ ID NO:2, (c-13) a CDRcomprising the amino acid sequence shown in SEQ ID NO:3, (d-13) a CDRcomprising the amino acid sequence shown in SEQ ID NO:77, (e-13) a CDRcomprising the amino acid sequence shown in SEQ ID NO:5, and (f-13) aCDR comprising the amino acid sequence shown in SEQ ID NO:6 Group 14(a-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:80,(b-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:3,(d-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:81,and (f-14) a CDR comprising the amino acid sequence shown in SEQ ID NO:6Group 15 (a-15) a CDR comprising the amino acid sequence shown in SEQ IDNO:1, (b-15) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-15) a CDR comprising the amino acid sequence shown in SEQ IDNO:66, (d-15) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-15) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-15) a CDR comprising the amino acid sequence shown in SEQID NO:6 Group 16 (a-16) a CDR comprising the amino acid sequence shownin SEQ ID NO:1, (b-16) a CDR comprising the amino acid sequence shown inSEQ ID NO:2, (c-16) a CDR comprising the amino acid sequence shown inSEQ ID NO:90, (d-16) a CDR comprising the amino acid sequence shown inSEQ ID NO:4, (e-16) a CDR comprising the amino acid sequence shown inSEQ ID NO:5, and (f-16) a CDR comprising the amino acid sequence shownin SEQ ID NO:6 Group 17 (a-17) a CDR comprising the amino acid sequenceshown in SEQ ID NO:1, (b-17) a CDR comprising the amino acid sequenceshown in SEQ ID NO:2, (c-17) a CDR comprising the amino acid sequenceshown in SEQ ID NO:52, (d-17) a CDR comprising the amino acid sequenceshown in SEQ ID NO:4, (e-17) a CDR comprising the amino acid sequenceshown in SEQ ID NO:5, and (f-17) a CDR comprising the amino acidsequence shown in SEQ ID NO:93 Group 18 (a-18) a CDR comprising theamino acid sequence shown in SEQ ID NO:98, (b-18) a CDR comprising theamino acid sequence shown in SEQ ID NO:2, (c-18) a CDR comprising theamino acid sequence shown in SEQ ID NO:3, (d-18) a CDR comprising theamino acid sequence shown in SEQ ID NO:4, (e-18) a CDR comprising theamino acid sequence shown in SEQ ID NO:5, and (f-18) a CDR comprisingthe amino acid sequence shown in SEQ ID NO:99 Group 19 (a-19) a CDRcomprising the amino acid sequence shown in SEQ ID NO:60, (b-19) a CDRcomprising the amino acid sequence shown in SEQ ID NO:2, (c-19) a CDRcomprising the amino acid sequence shown in SEQ ID NO:3, (d-19) a CDRcomprising the amino acid sequence shown in SEQ ID NO:4, (e-19) a CDRcomprising the amino acid sequence shown in SEQ ID NO:5, and (f-19) aCDR comprising the amino acid sequence shown in SEQ ID NO:99 Group 20(a-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:1,(b-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:2,(c-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:90,(d-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:4,(e-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:5,and (f-20) a CDR comprising the amino acid sequence shown in SEQ ID NO:6Group 21 (a-21) a CDR comprising the amino acid sequence shown in SEQ IDNO:1, (b-21) a CDR comprising the amino acid sequence shown in SEQ IDNO:2, (c-21) a CDR comprising the amino acid sequence shown in SEQ IDNO:3, (d-21) a CDR comprising the amino acid sequence shown in SEQ IDNO:4, (e-21) a CDR comprising the amino acid sequence shown in SEQ IDNO:5, and (f-21) a CDR comprising the amino acid sequence shown in SEQID NO:55 Group 22 (a-22) a CDR comprising the amino acid sequence shownin SEQ ID NO:1, (b-22) a CDR comprising the amino acid sequence shown inSEQ ID NO:2, (c-22) a CDR comprising the amino acid sequence shown inSEQ ID NO:66, (d-22) a CDR comprising the amino acid sequence shown inSEQ ID NO:110, (e-22) a CDR comprising the amino acid sequence shown inSEQ ID NO:5, and (f-22) a CDR comprising the amino acid sequence shownin SEQ ID NO:6 Group 23 (a-23) a CDR comprising the amino acid sequenceshown in SEQ ID NO:1, (b-23) a CDR comprising the amino acid sequenceshown in SEQ ID NO:2, (c-23) a CDR comprising the amino acid sequenceshown in SEQ ID NO:3, (d-23) a CDR comprising the amino acid sequenceshown in SEQ ID NO:4, (e-23) a CDR comprising the amino acid sequenceshown in SEQ ID NO:5, and (f-23) a CDR comprising the amino acidsequence shown in SEQ ID NO:115 Group 24 (a-24) a CDR comprising theamino acid sequence shown in SEQ ID NO:1, (b-24) a CDR comprising theamino acid sequence shown in SEQ ID NO:2, (c-24) a CDR comprising theamino acid sequence shown in SEQ ID NO:90, (d-24) a CDR comprising theamino acid sequence shown in SEQ ID NO:4, (e-24) a CDR comprising theamino acid sequence shown in SEQ ID NO:120, and (f-24) a CDR comprisingthe amino acid sequence shown in SEQ ID NO:6.
 8. The antibody of claim7, which comprises the combination of the light chain variable regionand the heavy chain variable region described in any one of thefollowing groups 25 to
 48. Group 25 (g-1) a light chain variable regioncomprising the above-identified CDRs (a-1) to (c-1); and (h-1) a heavychain variable region comprising the above-identified CDRs (d-1) to(f-1), Group 26 (g-2) a light chain variable region comprising theabove-identified CDRs (a-2) to (c-2); and (h-2) a heavy chain variableregion comprising the above-identified CDRs (d-2) to (f-2), Group 27(g-3) a light chain variable region comprising the above-identified CDRs(a-3) to (c-3); and (h-3) a heavy chain variable region comprising theabove-identified CDRs (d-3) to (f-3), Group 28 (g-4) a light chainvariable region comprising the above-identified CDRs (a-4) to (c-4); and(h-4) a heavy chain variable region comprising the above-identified CDRs(d-4) to (f-4), Group 29 (g-5) a light chain variable region comprisingthe above-identified CDRs (a-5) to (c-5); and (h-5) a heavy chainvariable region comprising the above-identified CDRs (d-5) to (f-5),Group 30 (g-6) a light chain variable region comprising theabove-identified CDRs (a-6) to (c-6); and (h-6) a heavy chain variableregion comprising the above-identified CDRs (d-6) to (f-6), Group 31(g-7) a light chain variable region comprising the above-identified CDRs(a-7) to (c-7); and (h-7) a heavy chain variable region comprising theabove-identified CDRs (d-7) to (f-7), Group 32 (g-6) a light chainvariable region comprising the above-identified CDRs (a-8) to (c-8); and(h-8) a heavy chain variable region comprising the above-identified CDRs(d-8) to (f-8), Group 33 (g-9) a light chain variable region comprisingthe above-identified CDRs (a-9) to (c-9); and (h-9) a heavy chainvariable region comprising the above-identified CDRs (d-9) to (f-9),Group 34 (g-10) a light chain variable region comprising theabove-identified CDRs (a-10) to (c-10); and (h-10) a heavy chainvariable region comprising the above-identified CDRs (d-10) to (f-10),Group 35 (g-11) a light chain variable region comprising theabove-identified CDRs (a-11) to (c-11); and (h-11) a heavy chainvariable region comprising the above-identified CDRs (d-11) to (f-11),Group 36 (g-12) a light chain variable region comprising theabove-identified CDRs (a-12) to (c-12); and (h-12) a heavy chainvariable region comprising the above-identified CDRs (d-12) to (f-12),Group 37 (g-13) a light chain variable region comprising theabove-identified CDRs (a-13) to (c-13); and (h-13) a heavy chainvariable region comprising the above-identified CDRs (d-13) to (f-13),Group 38 (g-14) a light chain variable region comprising theabove-identified CDRs (a-14) to (c-14); and (h-14) a heavy chainvariable region comprising the above-identified CDRs (d-14) to (f-14),Group 39 (g-15) a light chain variable region comprising theabove-identified CDRs (a-15) to (c-15); and (h-15) a heavy chainvariable region comprising the above-identified CDRs (d-15) to (f-15),Group 40 (g-16) a light chain variable region comprising theabove-identified CDRs (a-16) to (c-16); and (h-16) a heavy chainvariable region comprising the above-identified CDRs (d-16) to (f-16),Group 41 (g-17) a light chain variable region comprising theabove-identified CDRs (a-17) to (c-17); and (h-17) a heavy chainvariable region comprising the above-identified CDRs (d-17) to (f-17),Group 42 (g-18) a light chain variable region comprising theabove-identified CDRs (a-18) to (c-18); and (h-18) a heavy chainvariable region comprising the above-identified CDRs (d-18) to (f-18),Group 43 (g-19) a light chain variable region comprising theabove-identified CDRs (a-19) to (c-19); and (h-19) a heavy chainvariable region comprising the above-identified CDRs (d-19) to (f-19),Group 44 (g-20) a light chain variable region comprising theabove-identified CDRs (a-20) to (c-20); and (h-20) a heavy chainvariable region comprising the above-identified CDRs (d-20) to (f-20),Group 45 (g-21) a light chain variable region comprising theabove-identified CDRs (a-21) to (c-21); and (h-21) a heavy chainvariable region comprising the above-identified CDRs (d-21) to (f-21),Group 46 (g-22) a light chain variable region comprising theabove-identified CDRs (a-22) to (c-22); and (h-22) a heavy chainvariable region comprising the above-identified CDRs (d-22) to (f-22),Group 47 (g-23) a light chain variable region comprising theabove-identified CDRs (a-23) to (c-23); and (h-23) a heavy chainvariable region comprising the above-identified CDRs (d-23) to (f-23),Group 48 (g-24) a light chain variable region comprising theabove-identified CDRs (a-24) to (c-24); and (h-24) a heavy chainvariable region comprising the above-identified CDRs (d-24) to (f-24).9. The antibody of claim 8, which comprises the combination of the lightchain variable region and the heavy chain variable region described inany one of the following groups 49 to
 72. Group 49 (i-1) a light chainvariable region comprising the amino acid sequence shown in SEQ ID NO:8;and (j-1) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10, Group 50 (i-2) a light chain variable regioncomprising the amino acid sequence shown in SEQ ID NO:38; and (j-2) aheavy chain variable region comprising amino acid sequence shown in SEQID NO:10, Group 51 (i-3) a light chain variable region comprising theamino acid sequence shown in SEQ ID NO:41; and (j-3) a heavy chainvariable region comprising amino acid sequence shown in SEQ ID NO:10,Group 52 (i-4) a light chain variable region comprising the amino acidsequence shown in SEQ ID NO:44; and (j-4) a heavy chain variable regioncomprising amino acid sequence shown in SEQ ID NO:10, Group 53 (i-5) alight chain variable region comprising the amino acid sequence shown inSEQ ID NO:47; and (j-5) a heavy chain variable region comprising aminoacid sequence shown in SEQ ID NO:10, Group 54 (i-6) a light chainvariable region comprising amino acid sequence shown in SEQ ID NO:50;and (j-6) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10, Group 55 (i-7) a light chain variable regioncomprising amino acid sequence shown in SEQ ID NO:53; and (j-7) a heavychain variable region comprising amino acid sequence shown in SEQ IDNO:10, Group 56 (i-8) a light chain variable region comprising aminoacid sequence shown in SEQ ID NO:56; and (j-8) a heavy chain variableregion comprising amino acid sequence shown in SEQ ID NO:57, Group 57(i-9) a light chain variable region comprising amino acid sequence shownin SEQ ID NO:62; and (j-9) a heavy chain variable region comprisingamino acid sequence shown in SEQ ID NO:63, Group 58 (i-10) a light chainvariable region comprising amino acid sequence shown in SEQ ID NO:67;and (j-10) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:10, Group 59 (i-11) a light chain variable regioncomprising amino acid sequence shown in SEQ ID NO:71; and (j-11) a heavychain variable region comprising amino acid sequence shown in SEQ IDNO:72, Group 60 (i-12) a light chain variable region comprising aminoacid sequence shown in SEQ ID NO:75; and (j-12) a heavy chain variableregion comprising amino acid sequence shown in SEQ ID NO:10, Group 61(i-13) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:8; and (j-13) a heavy chain variable regioncomprising amino acid sequence shown in SEQ ID NO:78, Group 62 (i-14) alight chain variable region comprising amino acid sequence shown in SEQID NO:82; and (j-14) a heavy chain variable region comprising amino acidsequence shown in SEQ ID NO:83, Group 63 (i-15) a light chain variableregion comprising amino acid sequence shown in SEQ ID NO:86; and (j-15)a heavy chain variable region comprising amino acid sequence shown inSEQ ID NO:87, Group 64 (i-16) a light chain variable region comprisingamino acid sequence shown in SEQ ID NO:91; and (j-16) a heavy chainvariable region comprising amino acid sequence shown in SEQ ID NO:10,Group 65 (i-17) a light chain variable region comprising amino acidsequence shown in SEQ ID NO:94; and (j-17) a heavy chain variable regioncomprising amino acid sequence shown in SEQ ID NO:95, Group 66 (i-18) alight chain variable region comprising amino acid sequence shown in SEQID NO:100; and (j-18) a heavy chain variable region comprising aminoacid sequence shown in SEQ ID NO:101, Group 67 (i-19) a light chainvariable region comprising amino acid sequence shown in SEQ ID NO:104;and (j-19) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:101, Group 68 (i-20) a light chain variable regioncomprising amino acid sequence shown in SEQ ID NO:106; and (j-20) aheavy chain variable region comprising amino acid sequence shown in SEQID NO:10, Group 69 (i-21) a light chain variable region comprising aminoacid sequence shown in SEQ ID NO:108; and (j-21) a heavy chain variableregion comprising amino acid sequence shown in SEQ ID NO:57, Group 70(i-22) a light chain variable region comprising amino acid sequenceshown in SEQ ID NO:111; and (j-22) a heavy chain variable regioncomprising amino acid sequence shown in SEQ ID NO:112, Group 71 (i-23) alight chain variable region comprising amino acid sequence shown in SEQID NO:116; and (j-23) a heavy chain variable region comprising aminoacid sequence shown in SEQ ID NO:117, Group 72 (i-24) a light chainvariable region comprising amino acid sequence shown in SEQ ID NO:121;and (j-24) a heavy chain variable region comprising amino acid sequenceshown in SEQ ID NO:122.
 10. The antibody of claim 8, which comprises thecombination of the light chain and the heavy chain described in any oneof the following groups 73 to
 96. Group 73 (k-1) a light chaincomprising the amino acid sequence shown in SEQ ID NO:26; and (l-1) aheavy chain comprising the amino acid sequence shown in SEQ ID NO:28,Group 74 (k-2) a light chain comprising the amino acid sequence shown inSEQ ID NO:39; and (l-2) a heavy chain comprising the amino acid sequenceshown in SEQ ID NO:28, Group 75 (k-3) a light chain comprising the aminoacid sequence shown in SEQ ID NO:42; and (l-3) a heavy chain comprisingthe amino acid sequence shown in SEQ ID NO:28, Group 76 (k-4) a lightchain comprising the amino acid sequence shown in SEQ ID NO:45; and(l-4) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:28, Group 77 (k-5) a light chain comprising the amino acid sequenceshown in SEQ ID NO:48; and (l-5) a heavy chain comprising the amino acidsequence shown in SEQ ID NO:28, Group 78 (k-6) a light chain comprisingthe amino acid sequence shown in SEQ ID NO:51; and (l-6) a heavy chaincomprising the amino acid sequence shown in SEQ ID NO:28, Group 79 (k-7)a light chain comprising the amino acid sequence shown in SEQ ID NO:54;and (l-7) a heavy chain comprising the amino acid sequence shown in SEQID NO:28, Group 80 (k-8) a light chain comprising the amino acidsequence shown in SEQ ID NO:58; and (l-8) a heavy chain comprising theamino acid sequence shown in SEQ ID NO:59, Group 81 (k-9) a light chaincomprising the amino acid sequence shown in SEQ ID NO:64; and (l-9) aheavy chain comprising the amino acid sequence shown in SEQ ID NO:65,Group 82 (k-10) a light chain comprising the amino acid sequence shownin SEQ ID NO:68; and (l-10) a heavy chain comprising the amino acidsequence shown in SEQ ID NO:28, Group 83 (k-11) a light chain comprisingthe amino acid sequence shown in SEQ ID NO:73; and (l-11) a heavy chaincomprising the amino acid sequence is shown in SEQ ID NO:74, Group 84(k-12) a light chain comprising the amino acid sequence shown in SEQ IDNO:76; and (l-12) a heavy chain comprising the amino acid sequence shownin SEQ ID NO:28, Group 85 (k-13) a light chain comprising the amino acidsequence shown in SEQ ID NO:26; and (l-13) a heavy chain comprising theamino acid sequence shown in SEQ ID NO:79, Group 86 (k-14) a light chaincomprising the amino acid sequence shown in SEQ ID NO:84; and (l-14) aheavy chain comprising the amino acid sequence shown in SEQ ID NO:85,Group 87 (k-15) a light chain comprising the amino acid sequence shownin SEQ ID NO:88; and (l-15) a heavy chain comprising the amino acidsequence shown in SEQ ID NO:89, Group 88 (k-16) a light chain comprisingthe amino acid sequence shown in SEQ ID NO:92; and (l-16) a heavy chaincomprising the amino acid sequence shown in SEQ ID NO:28, Group 89(k-17) a light chain comprising the amino acid sequence shown in SEQ IDNO:96; and (l-17) a heavy chain comprising the amino acid sequence shownin SEQ ID NO:97, Group 90 (k-18) a light chain comprising the amino acidsequence shown in SEQ ID NO:102; and (l-18) a heavy chain comprising theamino acid sequence shown in SEQ ID NO:103, Group 91 (k-19) a lightchain comprising the amino acid sequence shown in SEQ ID NO:105; and(l-19) a heavy chain comprising the amino acid sequence shown in SEQ IDNO:103, Group 92 (k-20) a light chain comprising the amino acid sequenceshown in SEQ ID NO:107; and (l-20) a heavy chain comprising the aminoacid sequence shown in SEQ ID NO:28, Group 93 (k-21) a light chaincomprising the amino acid sequence shown in SEQ ID NO:109; and (l-21) aheavy chain comprising the amino acid sequence shown in SEQ ID NO:59,Group 94 (k-22) a light chain comprising the amino acid sequence shownin SEQ ID NO:113; and (l-22) a heavy chain comprising the amino acidsequence shown in SEQ ID NO:114, Group 95 (k-23) a light chaincomprising the amino acid sequence shown in SEQ ID NO:118; and (l-23) aheavy chain comprising the amino acid sequence shown in SEQ ID NO:119,Group 96 (k-24) a light chain comprising the amino acid sequence shownin SEQ ID NO:123; and (l-24) a heavy chain comprising the amino acidsequence shown in SEQ ID NO:124.
 11. An isolated antibody binding to thehuman CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with the antibody of claim
 7. 12. The antibody of claim11, which has a suppressive effect of T cell migration.
 13. An isolatedanti-human CD81 antibody, wherein the antibody comprises one or more ofCDRs described in any one of the groups 1 to 24 in claim 7 and binds tothe human CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with the antibody comprising all 6 CDRs described in saidgroup.
 14. An isolated anti-human CD81 antibody, wherein the antibodyhas a 90% sequence homology with any one of antibodies of claim 7 andbinds to the human CD81 having the amino acid sequence shown in SEQ IDNO:22 competitively with said antibody.
 15. An isolated anti-human CD81antibody comprising: (a-25) a CDR comprising the amino acid sequenceshown in SEQ ID NO:11; (b-25) a CDR comprising the amino acid sequenceshown in SEQ ID NO:12; (c-25) a CDR comprising the amino acid sequenceshown in SEQ ID NO:13; (d-25) a CDR comprising the amino acid sequenceshown in SEQ ID NO:14; (e-25) a CDR comprising the amino acid sequenceshown in SEQ ID NO:15; and (f-25) a CDR comprising the amino acidsequence shown in SEQ ID NO:16.
 16. The antibody of claim 15, whichcomprises: (g-25) a light chain variable region comprising theabove-identified CDRs (a-25) to (c-25); and (h-25) a heavy chainvariable region comprising the above-identified CDRs (d-25) to (f-25).17. The antibody of claim 16, which comprises: (i-25) a light chainvariable region comprising the amino acid sequence shown in SEQ IDNO:18; and (j-25) a heavy chain variable region comprising the aminoacid sequence shown in SEQ ID NO:20.
 18. The antibody of claim 16, whichcomprises: (k-25) a light chain comprising the amino acid sequence shownin SEQ ID NO:30; and (l-25) a heavy chain comprising the amino acidsequence shown in SEQ ID NO:32
 19. An isolated antibody binding to thehuman CD81 having the amino acid sequence shown in SEQ ID NO:22competitively with the antibody of claim
 15. 20. The antibody of claim19, which has a suppressive effect of T cell migration.
 21. An isolatedanti-human CD81 antibody, wherein the antibody comprises one or more ofCDRs in claim 15 and binds to the human CD81 having the amino acidsequence shown in SEQ ID NO:22 competitively with the antibody describedin claim
 15. 22. An isolated anti-human CD81 antibody, wherein theantibody has a 90% sequence homology with the antibody described inclaim 15 and binds to the human CD81 having the amino acid sequenceshown in SEQ ID NO:22 competitively with said antibody.
 23. The antibodyof claim 1 which is a humanized or human antibody.
 24. An isolatedpolynucleotide comprising a nucleotide sequence that encodes a heavychain variable region and a light chain variable region of the antibodyof claim
 1. 25. A combination of an isolated polynucleotide comprising anucleotide sequence that encodes a heavy chain variable region of theantibody of claim 7, and an isolated polynucleotide comprising anucleotide sequence that encodes the corresponding light chain variableregion of the antibody of claim
 7. 26. A combination of an isolatedpolynucleotide comprising a nucleotide sequence that encodes a heavychain variable region of the antibody of claim 15, and an isolatedpolynucleotide comprising a nucleotide sequence that encodes thecorresponding light chain variable region of the antibody of claim 15.27. An isolated polynucleotide comprising a nucleotide sequence thatencodes a heavy chain and a light chain of the antibody of claim
 1. 28.A combination of an isolated polynucleotide comprising a nucleotidesequence that encodes a heavy chain of the antibody of claim 7, and anisolated polynucleotide comprising a nucleotide sequence that encodesthe corresponding light chain of the antibody of claim
 7. 29. Acombination of an isolated polynucleotide comprising a nucleotidesequence that encodes a heavy chain of the antibody of claim 15, and anisolated polynucleotide comprising a nucleotide sequence that encodesthe corresponding light chain of the antibody of claim
 15. 30. Anexpression vector comprising the polynucleotide of claim
 24. 31. Arecombinant cell transformed with the expression vector of claim
 30. 32.A recombinant cell transformed with an expression vector comprising apolynucleotide comprising a nucleotide sequence that encodes the heavychain of the antibody of claim 7, and with an expression vectorcomprising a polynucleotide comprising a nucleotide sequence thatencodes the corresponding light chain of the antibody of claim
 7. 33. Arecombinant cell transformed with an expression vector comprising apolynucleotide comprising a nucleotide sequence that encodes the heavychain of the antibody of claim 15, and with an expression vectorcomprising a polynucleotide comprising a nucleotide sequence thatencodes the corresponding light chain of the antibody of claim
 15. 34. Amethod of producing an anti-human CD81 antibody, comprising culturingthe recombinant cell of claim 32, and recovering the antibody from theculture obtained.
 35. A method of producing an anti-human CD81 antibody,comprising culturing the recombinant cell of claim 33, and recoveringthe antibody from the culture obtained.
 36. A pharmaceutical compositioncomprising the antibody of claim
 1. 37. A method for the prophylaxis,improvement or treatment of a disease selected from inflammatory boweldisease, multiple sclerosis, psoriasis and hematological cancer,comprising administering to a subject an effective amount of theantibody of claim 1.